Intrathecal and Intratumoral Superantigens to Treat Malignant Disease

ABSTRACT

The presence of tumor nodules in organs often results in serious clinical manifestations and the permeation by cancer cells of sheaths surrounding organs often produces clinical manifestations of pleural effusion, ascites or cerebral edema. The present invention addresses this problem by providing a method for treating minors comprising (a) intratumoral administration of a superantigen and/or (b) intrathecal or intracavitary administration of a superantigen directly into the sheath. Intratumoral superantigen results in significant and sustained reduction of the tumor size. Intrathecal administration produces significant sustained reduction of the fluid accumulation associated with clinical improvement and prolonged survival. Useful superantigen compositions for intrathecal and intratumoral injection include tumoricidally effective homologues, fragments and fusion proteins of native superantigens. Also disclosed is combined therapy that includes intratumoral or intrathecal superantigen compositions in combination with (i) intratumoral low, non-toxic doses of one or more chemotherapeutic drugs or (ii) systemic chemotherapy at reduced and non-toxic doses of chemotherapeutic drugs.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention in the fields of immunology and medicine is directed to amethod for treating a category of neoplastic diseases that are manifestin sheaths surrounding organs (intrathecal) by administering tumoricidalsuperantigens such as bacterial enterotoxins and various biologicallyactive derivatives thereof.

2. Description of the Background Art

Staphylococcal enterotoxins (“SE's”) are representative of a family ofproteins known as “superantigens” (SAg)—the most powerful T lymphocytemitogens known. They can activate between about 5 and about 30% or thetotal T cell population compared to the activation of 0.01% or fewer Tcells by conventional antigens. Moreover, these enterotoxins elicitstrong polyclonal proliferative responses at concentrations about10³-fold lower than other T cell mitogens. The most potent SE on a perweight basis, Staphylococcal enterotoxin A (SEA), stimulates human Tcell proliferation (measured as DNA synthesis) at concentrations of aslow as 10⁻¹³-10⁻¹⁶M.

SAg-activated T cells produce a variety of cytokines, includinginterferon-γ (IFNγ), various interleukins and tumor necrosis factor-α(TNFα) (Dohlsten et al., Int. J. Cancer 54:482-488 (1993).

SAgs also stimulate other cell populations involved in innate andadaptive immunity and contribute to anti-tumor immunity. For example,SE's engage the variable (V) region of the T cell receptor (TCR) chainon the exposed face of the pleated sheet and the sides of the MHC classII molecule (Kiting B L et al., Adv Immunol. 1993; 54:99-166). SAgsaugment T_(H)1 cytokine response by CD4+ cells while also activatingcells of the NK, NKT and γ/δ T cell lineages. Cytotoxic action of NKcells is augmented by the IFNγ produced by SAg activated T cells (Moritaet al., Immunity 14:331-44. (2001) D'Orazio J A et al., J Immunol.154:1014-23 (1995).

SAgs induce tumor killing in vivo when given alone or when conjugated totumor-specific antibodies (Terman U.S. Pat. No. 6,221,351; Dohlsten U.S.Pat. No. 5,858,363). They are also effective when employed ex vivo toinduce the generation of tumor sensitized T cells that are thenadministered in the “adoptive therapy” of (e.g., MCA 205/207) tumors(Shu et al. J Immunol. 152: 1277-1288 (1994). SAg-transfected tumorcells can reduce metastatic disease in an established murine mammarycarcinoma model (Pulaski et al., Cancer Res. 60: 2710-5 (2000).

In addition to these biological activities, the SE's share commonphysicochemical properties. They are heat stable, trypsin-resistant, andsoluble in water and salt solutions, have similar sedimentationcoefficients, diffusion constants, partial specific volumes, isoelectricpoints, and extinction coefficients. Prior to more recent discoveries ofadditional SE's, earlier-described SEs were divided into fiveserological types designated SEA, Staphylococcal enterotoxin B (SEB),Staphylococcal enterotoxin C (SEC), Staphylococcal enterotoxin D (SED)and Staphylococcal enterotoxin E (SEE), which exhibit strikingstructural similarities.

An SE is a single polypeptide chain of about 30 kDa. All SEs have acharacteristic disulfide loop near the middle of the chain. SEA is aflat monomer consisting or 233 amino acids divided into two domains:domain I comprising residues 31-116 and domain II comprising residues117-233 together with the amino tail of residues 1-30. The biologicallyactive regions of the proteins are evolutionarily conserved and show arelatively higher degree of sequence homology/similarity. One region ofstriking amino acid sequence homology between SEA, SEB, SEC, SED, andSEE is located immediately on the C-terminal side of Cys-106 (in SEA).This conserved region is thought to be responsible for T cellactivation. A second conserved homology region, at about residue 147, isbelieved to be responsible for emetic activity. This emesis-inducingregion can be deleted from SE's through genetic engineering; suchmodified SE's are also useful therapeutics in accordance with thisinvention.

Sequence analysis of SEs and comparison with other bacterial toxinsrevealed SEA, SEB, SEC, SED, Staphylococcal toxic shock-associated toxin(TSST-1, also known as SEF), and the Streptococcal pyrogenic exotoxins(SpE's) share considerable nucleic acid and amino acid sequencesimilarity (Betley et al., J. Bacteriol. 170: 34-41 (1988)). Thus, theSEs belong to a family of evolutionarily related proteins.

SEs bind to MHC class II molecules and TCRs in a manner quite distinctfrom conventional antigens. SEs engage the V region of the TCR β chain(Vβ region) on an exposed face of the β pleated sheet. SEs engage the“sides” of MHC class II molecule rather than engaging the groove as doconventional antigens. In contrast to SEB and the SEC, which bind onlyto the MHC class II α chain, SEA, as well as SEE and SED, also interactwith the MHC class II α chain in a zinc-dependent manner (Fraser J D etal., Proc. Natl. Acad. Sci. 89:5507-11 (1992)).

T cell recognition of SAgs, such as SEs, via the TCR Vβ region isindependent of other TCR components and diversity elements. Single aminoacid positions and regions important for SAg-TCR interactions have beendefined. These residues are located in the vicinity of the shallowcavity formed between the two SE domains. (Lavoie P M et al., Immunol.Rev. 168: 257-269 (1999). Substitution of amino acid residue Asn23 inSEB by Ala has demonstrated the importance of this position in SEB/TCRinteractions. This particular residue is conserved among all of the SE'sand may constitute a common anchor position for SE interaction with TCRstructures. Amino acid residues in positions 60-64 of SEA contribute tothe TCR interaction as do the Cys residues forming the intramoleculardisulfide bridge (Kappler J et al., J. Exp. Med. 175 387-96 (1992)). ForSEC2 and SEC3, the key points of interaction in the TCR Vβ region arelocated in the CDR1, CDR2 and HRV4 regions of the TCR Vβ3 chain(Deringer J R et al., Mol. Microbiol. 22: 523-534 (1996)). Hence,multiple and highly variable parts of the Vβ region contribute to theformation of the TCRs SE binding site.

Thus far, no single, linear consensus motif in the TCR Vβ displaying ahigh affinity interaction with particular enterotoxins has beenidentified. A significant contribution of the TCRα chain in SE-TCRrecognition is acknowledged (Smith et al., J. Immunol. 149: 887-896(1992)). It is apparently the distinctive binding characteristics of SEswhich bypass the highly variable parts of the MHC class II and TCRmolecules that endows SEs with their ability to activate such a highfrequency of T cells and cause massive proliferation, cytokine inductionand cytotoxic T cell generation. These properties are shared by otherproteins produced by various infectious agents. Together, these proteinsform a well recognized family of molecules, Sags, because of theiraforementioned biological effects.

Mycoplasmal, viral, and other bacterial proteins are SAgs. In additionto SEs and SpEs, examples include Yersinia pseudotuberculosis mitogenicprotein (“YPM”), and Clostridium perfringens toxin A. All SAgs activateT cells without a requirement for conventional antigen processing, andthe responding T cells do not respond in a conventional MHC restrictedmanner. As noted, SAgs bind to and evoke responses from all T cellsexpressing certain TCR Vβ gene products independently of other TCRstructures. CD4⁻ CD8⁻ TCR α/β T cells and γ/δ T cells all respond toSAgs by proliferation, production of T_(H)1 cytokines and generation ofcytotoxic activity.

Native SEs are known to induce anti-tumor effects. Administration of SEBproduced antitumor effects against established tumors in two animalspecies, rabbits and mice, with tumors of five different histologictypes: rabbit VX-2 carcinoma (Terman et al., U.S. Pat. No. 6,126,945;Terman, U.S. Pat. No. 6,340,461), murine CL 62 melanomas (Penna C. etal., Cancer Res. 54: 2738-2743 (1994)), murine A/20 lymphoma (Kalland T.Declaration in U.S. Ser. No. 07/689/799 (1992)), murine PRO4Lfibrosarcoma (Newell et al., Proc Natl. Acad. Sci. 88: 1074-1079 (1991))and human SW 620 colon carcinoma (Dohlsten et al., Eur. J. Immunol. 21:1229-1233 (1991)). In these studies, parenterally-administered SEBinduced objective anti-tumor effects at primary and metastatic sites.SEB was used ex vivo to stimulate a population of T cells pre-exposed totumor, which, upon re-infusion into host animals with establishedpulmonary metastases, induced a substantial reduction of metastases. SEBactivated T cell anti-tumor effect was specific for the immunizingtumor; the SEB stimulated T cells produced IFNγ which was thought to bean important mediator of the anti-tumor effect (Shu S et al., J.Immunol. 152: 1277-88 (1994)).

Fusion polypeptides comprising SEA fused to a tumor specific monoclonalantibody (mAb), designated “SEA-mAb,” induced tumoricidal responses inthe murine B16 melanoma model (Dohlsten M et al., Proc Natl Acad Sci91:8945-9 (1994); Dohlsten M et al., Proc. Natl. Acad. Sci. 88:9287-91(1991).

Because native SEA alone was found to be ineffective in such models,Dohlsten and colleagues (U.S. Pat. No. 5,858,363) stated that native SAgwould be of “low value” particularly against MHC class II-negativetumors. When the fusion polypeptides SEA-mAb and one comprising a mutantSEA, “D227A-mAb” (these authors used ‘MoAb’ rather than ‘mAb’ forabbreviating ‘monoclonal antibody’) were given to human patients withadvanced colon carcinoma, SEs reacted with preexisting “natural”SE-specific antibodies which diminished the antitumor effects in vivo.Following additional doses of SE-mAb preparations, the anti-SE antibodylevels increased significantly (Giantonio et al., J. Clin. Oncol.15:1994-2007 (1997); Alpaugh et al., Clin. Cancer Res. 4:1903-14 (1998);Persson et al., Adv. Drug Del. Res. 31: 143-152 (1998)). To date,efforts to overcome this problem have met with only partial success.

Tumors in Sheaths Encasing Organs

The appearance of tumors in sheaths (“theca”) encasing organs oftenresults in production and accumulation of large volumes of fluid in theorgans' sheath. Examples include (1) pleural effusion due to fluid inthe pleural sheath surrounding the lung, (2) ascites originating fromfluid accumulating in the peritoneal membrane and (3) cerebral edema dueto metastatic carcinomatosis of the meninges. Such effusions and fluidaccumulations generally develop at an advanced stage of the disease.Malignant pleural effusion (“MPE”) is the prototype of this condition.In the United States and Western Europe, 300,000 new cases of malignantpleural effusion are diagnosed annually (Antony V B et al., Eur. Respir.J. 18:402-419 (2001). This condition is caused by different types oftumors: lung cancer (35%), breast cancer (25%), lymphoma (10%), unknownprimary malignancy (30%). It is the presenting manifestation in 10-50%of all cancers. When first evaluated, about 15% of lung cancer patientsexhibit a pleural effusion. Fifty percent of cancer patients develop MPEat some point in their disease process, and up to 75% of MPE cases aresymptomatic from their effusions upon presentation. The appearance of apleural effusion in non small cell lung cancer (NSCLC) signifies StageMb or Stage IV and a poor prognosis with a median survival on the orderto 2-3 months (1, 7-11). In this group, no significant difference insurvival were observed between those with cytologically positive andnegative effusions (12). Although most of these patients are symptomaticand/or disabled from their effusions, they are not surgical candidates.They are usually offered palliative treatment with chemical pleurodesis.

Malignant ascites is associated with 30-50% of ovarian tumors.Endometrial, breast, colonic, gastric and pancreatic carcinomas make upmore than 80% or the tumors associated with intra-abdominal seeding oftumor cells and ascites formation. Ascites may be the presentingmanifestation in 4-69% of cases.

The major therapies for MPE include talc poudrage, talc slurry,doxycycline and bleomycin instillation (Veena et al. Am J. Crit. CareMed. 162: 1987-2001 (2000)). These therapies require 3-12 days ofhospitalization with EKG and oximetry monitoring. A chest tube isinserted, and the therapeutic agent is infused and allowed to distributeover the pleural membranes. The chest tube is then connected to closednegative-pressure water seal drainage until pleural fluid volume dropsbelow 100 ml/24 hours. Respiratory therapy is usually given at leastonce daily.

Talc poudrage requires the use of operating room and general anesthesiafor thoracostomy and talc insufflation, followed by recovery roomobservation. Talc induces respiratory complications in up to 33% ofpatients and acute respiratory distress and hypoxemia in 10% ofpatients. Response rates to bleomycin and doxycycline range between 50%and 70%, respectively and both require continuous chest tube drainageuntil the output is below 100 ml/24 hours. Indwelling pleural cathetersfor drainage and/or injection of a pleurodesis agent are an additionaloption (7,8); however, the catheter requires surgical placement followedby intermittent drainage of effusion fluid at home by the patient or acaregiver.

Intrapleurally administered agents or modalities that include (a)chemotherapeutic agents such as Cisplatin, Cytarabine, Doxorubicinfluorouracil, etoposide, and mitomycin C, (b) radiation and (c)biotherapeutic agents such as IL-2, various interferons, and bacteriallyderived immunostimulatory agents such as Corynebacterium parvum havebeen ineffective against MPEs. Thoracentesis or chest tube drainagealone results in recurrence rates of 98% and 85% respectively within 30days. Intraperitoneal cisplatin and etoposide has produced a completeresponse rate of 30% of malignant ascites. However the only randomizedstudy has failed to show any benefit for intraperitoneal therapy overconventional intravenous chemotherapy in the initial management of stageII C to IV ovarian cancer. No definitive success of various biologicagents, e.g., IFN-α, β, and γ, TNFα or IL-2 has been reported.

The present invention overcomes these deficiencies in the treatment ofMPE by providing a new therapeutic approach to this manifestation ofcancer. Unlike existing therapies, The present invention is carried outentirely in an outpatient setting and requires no hospitalization at acost several hundred percent below that of existing therapy. Major costsof the other therapies originating from hospitalization, chest tubeinsertion, operating and recovery room expense, respiratory therapy andin-hospital chest tube drainage, are eliminated

Intratumoral SAg Therapy

Prior to the present invention, therapeutic uses of SAgs have beenlimited to systemic administration. To improve the ability SAgs tolocalize to a tumor, investigators have taken two approaches. In oneapproach, they have produced mutant SAg molecules with reduced bindingto MHC class II molecules (Hansson J et al., Proc. Nail Acad. Acad Sci.USA 94: 2489-94 (1997)). In the second approach, they have conjugated atumor specific antibody to the SAg (Dohlsten M et al., Proc Nail AcadSci USA 91:8945-9 (1994); Dohlsten M et al., Proc Nail Acad Sci USA88:9287-91 (1991)). However, because SAg-specific antibodies are foundin all humans, these engineered molecules, rather than localizing totumors, are more likely to be directed to reticuloendothelial tissueswhere they are degraded and eliminated. The researchers cited aboveexpressly asserted (U.S. Pat. No. 5,858,363 that native SAgs would be of“low value” for such antitumor therapy because cells of most clinicallyimportant tumors do not express MEM class II molecules. It is thereforeevident that those working in this field, led by the investigators citedabove, did not envision the use of the SAgs by intratumoraladministration. In contrast to systemic administration, intratumoraldelivery of a SAg would not require alteration of the native moleculeand, as conceived by the present inventor, the presence of naturalantibodies throughout the body can actually assistintratumorally-administered SAgs in evoking a tumoricidal response.

SUMMARY OF THE INVENTION

The present invention provides a method for treating malignant pleuraleffusion, ascites, pericardial effusion and meningeal carcinomatosis by“intrathecal” (defined below) administration of an effective amount of aSAg into the pleural space, peritoneum, pericardium, and subarachnoidspace. The present invention contemplates the use of any SAg, includingbut not limited to staphylococcal enterotoxins (“SE”) A, B, C, D, E, F,G H, I, J, K, L, M, streptococcal pyrogenic exotoxins (SpE's), Yersiniapseudotuberculosis SAg, Mycoplasma arthritides SAg, and C. perfringensexotoxin, administered by injection, infusion or instillation directlyinto a cavity or space (thecum) surrounding an organ or body region inwhich a tumor is present or is causing fluid accumulation.

Such spaces include the pleural space, peritoneum, subarachnoid space ordural space, or pericardial space. The generic term for administrationinto a sheath encasing an organ is termed “intrathecal,” defined inDorland's Medical Dictionary 29^(th) Edition, WB Saunders (2000) andStedman's Medical Dictionary, 27^(th) Edition, Lippincott, Williams &Wilkins (2000) as meaning “within a sheath.” As used herein, this termis intended to be broader than a more commonly used definition which islimited to intracranial spaces.

Previous publications disclose administration of SEs to a host withcancer via subcutaneous or intravenous injection or infusion. (See, forexample, U.S. Pat. No. 6,126,945.) Other document disclose theadministration of SAgs “locally or systemically” (U.S. Pat. No.6,197,299; U.S. Pat. No. 5,858,363) or in adjuvants with slow release(U.S. Pat. No. 6,126,945, by the present inventor). However, the priorart does not disclose administration of a SAg intrathecally or morespecifically, intrapleurally and intraperitoneally, to treat malignantpleural effusions and ascites. Nor does the prior art disclose theadministration of a SAg intratumorally. SAgs in native form orconjugated to a tumor targeting agent such as monoclonal antibodies ortheir fragments (“mAbs”) have been used to treat cancer in animals andhumans (Hansson et al., Proc. Natl. Acad. Sci. 94:2489 (1997)). In allof these instances, the SAg or SAg-mAb conjugate/fusion wereadministered intravenously by injection or infusion.

When administered in this way SAgs or SAg conjugated to tumor specificantibodies (SAg-mAb fusion proteins) do not reach their targets ineffective concentrations for two reasons. First, the SAgs areneutralized rapidly by “natural” neutralizing SAg-specific antibodies.(Giantonio et al., supra; Alpaugh et al supra; Persson et al., supra).Second, SAg-mAb fusion proteins bind to cells present in the circulationthat express MHC class II proteins. One approach to overcoming theseobstacles was to mutate the SAg to reduce its affinity for MHC class II.This has met with only partial success (Hansson et al supra).

The present invention obviates this obstacle to a large extent byadministering a SAg (including a fragment, homologue or fusion partner),intrathecally, into sheaths encasing the organs which themselves areseeded with tumor or directly into tumor site(s). In this case, anypre-existing or induced anti-SAg antibodies may actually assist theintrathecally or intratumorally administered SAg in promoting tumorkilling by binding to the SAg after it has localized to tumor cellsurfaces.

The present invention covers compositions of SAg or SAg homologuesconsisting of amino acid substitution and deletion variants (mutants),additions (e.g., fusion proteins) and fragments with Z values >10 whenthe sequence is compared to a native SAg using the FASTA/FASTP programsand Monte Carlo analysis. These composition are injected, instilled orinfused intrapleurally or intraperitoneally into a patient withmalignant pleural effusions and/or ascites, respectively orintratumorally into tumor site(s) and induce a tumoricidal response. TheSAg composition is preferably administered after partial or completedrainage of the fluid from the sheath as for example in pleuraleffusions via thoracentesis and ascites via paracentesis. However, theSAg composition may also be administered directly into an undrainedspace containing the effusion, ascites and/or carcinomatosis. Theinvention also contemplates the use of the nucleic acid counterparts ofthe native SAgs and homologues as useful for the same indications as thepolypeptide form of the molecule.

To enhance the effectiveness and specificity of the SAg, it or abiologically active fragment or homologue may be fused to anotherprotein such as (1) a tumor specific antibody, or an antigen bindingfragment of such an antibody, such as an F(ab′)₂, Fv or Fd fragment,which antibody is specific for an epitope expressed on the tumor or (b)a receptor ligand specific for any receptors selectively orpreferentially expressed on tumor cells. The fusion partner can also bea powerful costimulant such as OX-40 or 4-1BB1 which enhances the Tcells proliferative response to the SAg or a “Coaguligand” whichpromotes coagulation in the tumor vasculature.

The SAg composition is administered once every 3 to 10 days, preferablyonce weekly, and this schedule is continued until there is nore-accumulation of the effusion or ascites or reduction in the size ofthe tumor mass being injected. Three such treatments may suffice forintrathecal administration although this is an average; the number oftreatments may varying from 1-6 or even higher. For intrathecaladministration, the SAg composition is preferentially administeredimmediately after removal of pleural fluid via thoracentesis. Unlike theother therapies for malignant pleural effusions, the present method iscarried out entirely in an outpatient setting and requires nohospitalization, chest tube insertion, use of the operating room orrecovery room, respiratory therapy or in-hospital chest tube drainage.In contrast to the conventional treatment for MPE noted above,instillation of the SAg composition into the pleural space has aresponse rate of nearly 100%. Unlike talc therapy in which up to 10% ofcases may experience acute respiratory distress syndrome, the presentSAg therapeutic method has not induced any significant morbidity. Hence,this invention offers decided advantages of effectiveness, safety andconvenience over the prior art.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows Kaplan-Meir survival curve of the SEC-treated groupcompared to current historic control group of 21 patients with MPE fromNSCLC treated at UCSD from 1993-1998 with talc insufflation showing asignificantly prolonged survival in the SEC-treated group compared tothe control group (p=0.0096).

FIG. 2 shows Kaplan-Meir survival curve of all patients in SEC-treatedgroup with KPS range of 30-60 compared to a subset of patients from thecontrol group in FIG. 1A with the same KPS range 30-60. Median KPS forthe SEC and control groups were 45 and 43 respectively (p=0.8) yet theSEC-treated group showed a significantly prolonged survival (p=0.014).

DESCRIPTION OF THE PREFERRED EMBODIMENTS Production and Isolation ofSuperantigens

The SAgs disclosed herein are prepared by either biochemical isolation,or, preferably by recombinant methods. The following SAgs, includingtheir sequences and biological activities have been known for a numberof years. Studies of these SAgs are found throughout the biomedicalliterature. For, biochemical and recombinant preparation of these SAgs,see the following references: Borst, D W et al., Infect. Immun. 61:5421-5425 (1993); Couch, J L et al., J. Bacteria 170: 2954-2960 (1988);Jones, C L et al., J. Bacteriol. 166: 29-33 (1986); Bayles K W et al.,J. Bacteria 171: 4799-4806 (1989); Blomster-Hautamaa, D A et al., J.Biol. Chem. 261:15783-15786 (1986); Johnson, L P et al., Mol. Gen.Genet. 203, 354-356 (1986); Bohach G A et al., Infect. Immun. 55:428-433 (1987); Iandolo J J et al., Methods Enzymol 165:43-52 (1988);Spero L et al., Methods Enzymol 78(Pt A):331-6 (1981); Blomster-HautamaaD A, Methods Enzymol 165: 37-43 (1988); Iandolo J J Ann. Rev. Microbial.43: 375-402 (1989); U.S. Pat. No. 6,126,945 and U.S. provisional patentapplication 60/389,366 filed Jun. 15, 2002. These references and thereferences cited therein are hereby incorporated by reference in theirentirety.

These SAgs are Staphylococcal enterotoxin A (SEA), Staphylococcalenterotoxin B (SEB), Staphylococcal enterotoxin C (SEC—actually threedifferent proteins, SEC1, SEC2 and SEC3)), Staphylococcal enterotoxin D(SED), Staphylococcal enterotoxin E (SEE) and toxic shock syndrometoxin-1 (TSST-1) (U.S. Pat. No. 6,126,945 and U.S. provisional patentapplication 60/389,366 filed Jun. 15, 2002, and the references citedtherein). The amino acids sequences of the above group of native(wild-type) SAgs is provided below:

SEA (Huang, I. Y. et al., J. Biol. Chem. 262: 7006-7013 (1987)) [SEQ IDNO: 1] 1 SEKSEEINEK DLRKKSELQG TAGNKQIY YYNEKAKTEN KESHDQFLQH TILFKGFFTD61 HSWYNDLLVD FDSKDIVDKY KGKKVDLYGA YYGYQCAGGT PNKTACMYGG VTLHDNNRLT 121EEKKVPINLW LDGKQNTVPL ETVKTNKKNV TVQELDLQAR RYLQEKYNLY NSDVFDGKVQ 181RGLIVFHTST EPSVNYDLFG AQGQYSNTLL RIYRDNKSIN SENMHIDIYL YTS SEB(Papageorgiou, A. C. et al. J. Mol. Biol. 277: 61-79 (1998)) [SEQ ID NO:2] 1 ESQPDPKPDE LHKSSKFTGL MENMKVLYDD NHVSAINVKS IDQFLYFDLI YSIKDTKLGN61 YDNVRVEFKN KDLADKYKDK YVDVFGANYY YQCYFSKKTN DINSHQTDKR KTCMYGGVTE 121HNGNQLDKYR SITVRVFEDG KNLLSFDVQT NKKKVTAQEL DYLTRHYLVK NKKLYEFNNS 181PYETGYIKFI ENENSFWYDM MPAPGDKFDQ SKYLMMYNDN KMVDSKDVKI EVYLTTKK SEC1(Bohach, G. A. et al., Mol. Gen. Genet. 209: 15-20 (1987)) [SEQ ID NO:3] 1 MNKSRFISCV ILIFALILVL FTPNVLAESQ PDPTPDELHK ASKFTGLMEN MKVLYDDHYV61 SATKVKSVDK FLAHDLIYNI SDKKLKNYDK VKTELLNEGL AKKYKDEVVD VYGSNYYVNC 121YFSSKDNVGK VTGGKTCMYG GITKHEGNHF DNGNLQNVLI RVYENKRNTI SFEVQTDKKS 181VTAQELDIKA RNFLINKKNL YEFNSSPYET GYIKFIENNG NTFWYDMMPA PGDKFDQSKY SEC2(Papageorgiou, A. C., et al., Structure 3: 769-779 (1995)) [SEQ ID NO:4] 1 ESQPDPTPDE LHKSSEFTGT MGNMKYLYDD HYVSATKVMS VDKFLAHDLI YNISDKKLKN61 YDKVKTELLN EDLAKKYKDE VVDVYGSNYY VNCYFSSKDN VGKVTGGKTC MYGGITKHEG 121NHFDNGNLQN VLIRVYENKR NTISFEVQTD KKSVTAQELD IKARNFLINK KNLYEFNSSP 181YETGYIKFIE NNGNTFWYDN MPAPGDKFDQ SKYLMMYNDN KTVDSKSVKI EVHLTTKNG SEC3(Hovde, C. J. et al., Mol. Gen. Genet. 220: 329-333 (1990)) [SEQ ID NO:5] 1 MYKRLFISRV ILIFALILVI STPNVLAESQ PDPMPDDLHK SSEFTGTMGN MKYLYDDHYV61 SATKVKSVDK FLAHDLIYNI SDKKLKNYDK VKTELLNEDL AKKYKDEVVD VYGSNYYVNC 121YFSSKDNVGK VTGGKTCMYG GITKHEGNHF DNGNLQNVLV RVYENKRNTT SFEVQTDKKS 181VTAQELDIKA RNFLINKKNL YEFNSSPYET GYIKFIENNG NTFWYDMMPA PGDKFDQSKY 241LMMYNDNKTV DSKSVKIEVH LTTKNG SED (Bayles, K. W. et al., J. Bacteriol.171: 4799-4806 (1989)) [SEQ ID NO: 6] 1 MKKFNILIAL LFFTSLVTSP LNVKANENIDSVKEKELHKK SELSSTALNN MKHSYADKNP 61 IIGENKSTGD QFLENTLLYK KFFTDLINFEDLLINFNSKE MAQHFKSKNV DVYPIRYSIN 121 CYGGEIDRTA CTYGGVTPHE GNKLKERKKIPINLWINGVQ KEVSLDKVQT DKKNVTVQEL 181 DAQARRYLQK DLKLYNNDTL GGKIQRGKIEFDSSDGSKVS YDLFDVKGDF PEKQLRIYSD 241 NKTLSTEHLH IDIYLYEK SEE (Couch. J.L. et, al. J. Bacteriol. 170: 2954-2960 (1988)) [SEQ ID NO: 7] 1MKKTAFILLL FIALTLTTSP LVNGSEKSEE INEKDLRKKS ELQRNALSNL RQIYYYNEKA 61ITENKESDDQ FLENTLLFKG FFTGHPWYND LLVDLGSKDA TNKYKGKKVD LYGAYYGYQC 121AGGTPNKTAC MYGGVTLHDN NRLTEEKKVP INLWIDGKQT TVPIDKVKTS KKEVTVQELD 181LQARHYLHGK FGLYNSDSFG GKVQRGLIVF HSSEGSTVSY DLFDAQGQYP DTLLRIYRDN 241KTINSENLHI DLYLYTT TSST-1 (Prasad, G. S. et al., Protein Sci. 6:1220-1227 (1997)) [SEQ ID NO: 8] 1 MNKKLLMNFF IVSPLLLATT ATDFTPVPLSSNQIIKTAKA STNDNIKDLL DWYSSGSDTF 61 TNSEVLDNSL GSMRIKNTDG SISLIIFPSPYYSPAFTKGE KVDLNTKRTK KSQHTSEGTY 121 IHFQISGVTN TEKLPTPIEL PLKVKVHGKDSPLKYGPKFD KKQLAISTLD FEIRHQLTQI 181 HGLYRSSDKT GGYWKITMND GSTYQSDLSKKFEYNTEKPP INIDEIKTIE AEIN

The sections which follow discuss SAgs which have been discovered andcharacterized more recently.

Staphylococcal Enterotoxins SEG, SEH, SEI, SEJ, SEK, SEL, SEM

New Staphylococcal enterotoxins G, H, 1, J, K, L and M (SEG, SEH, SET,SEJ, SEK, SEL, SEM; abbreviated below as “SEG-SEM”) were described inJarraud, S. et al., J. Immunol. 166: 669-677 (2001); Jarraud S et al.,J. Clin. Microbial. 37: 2446-2449 (1999) and Munson, S H et al., Infect.Immun. 66:3337-3345 (1998). SEG-SEM show SAgic activity and are capableof inducing tumoricidal effects. The homology of these SE's to thebetter known SE's in the family ranges from 27-64%. Each inducesselective expansion of TCR Vβ subsets Thus, these SEs retain thecharacteristics of T cell activation and Vβ usage common to all theother SE's.

SEG and SEH of this group and other enterotoxins including SPEA, SPEC,SPEG, SPEH, SME-Z, SME-Z₂, (see below) utilize zinc as part of highaffinity MHC class II receptor. Amino acid substitution(s) at thehigh-affinity, zinc-dependent class II binding site are created toreduce their affinity for MHC class II molecules.

Jarraud S et al., 2001, supra, discloses methods used to identify andcharacterize SEG-SEM, and for cloning and recombinant expression ofthese proteins. These investigators have used a number of TCR-specificmAbs (Vfβ specificity indicated in brackets) for flow cytometricanalysis: E2.2E7.2 (Vβ2), LE89 (VP), IMMU157 (Vfβ5.1), 3D11 (Vβ5.3),CRI304.3 (Vβ6.2), 3G5D15 (Vβ7), 56C5.2 (Vβ8.1/8.2), FIN9 (Vβ9), C21(Vβ11), S511 (Vβ12), IMMU1222 (Vβ13.1), JIJ74 (Vβ13.6), CAS1.1.13(Vβ14), Tamaya1.2 (Vβ16), E17.5F3 (Vβ17), βA62.6 (Vβ18), ELL1.4 (Vβ20),IG125 (Vβ21.3), IMMU546 (Vβ22), and HUT78.1 (Vβ23).

Jarraud S et al., 2001, supra, indicates that the seven genes andpseudogenes composing the egc (enterotoxin gene cluster) operon areco-transcribed. The association of related co-transcribed genessuggested that the resulting peptides might have complementary effectson the host's immune response. One hypothesis was that generecombination created new SE variants differing by their SAg activityprofiles. Purified recombinant SEL, SEM, SEI, SEK, and SEGL29P (a mutantof SEL) were expressed in E. coli and analyzed. Recombinant SEL SEM,SEI, and SEK consistently induced selective expansion of distinctsubpopulations of T cells expressing particular Vβ genes. By contrast.SEGL29P failed to trigger expansion of any of 23 Vβ subsets, and theL29P mutation accounted for the complete loss of SAg activity (althoughthis mutation did not induce a major conformational change). It isbelieved that this substitution mutation located at a position crucialfor proper SAg/MHC II interaction.

Flow cytometry revealed preferential expansion of CD4 T cells in SET andSEM cultures. By contrast, the CD4/CD8 ratios in SEK-, SEL-, andSEG-stimulated T cell lines were close to those in fresh PBL. Overall,TCR repertoire analysis confirm the SAgic nature of SEG-SEM.

The amino acid sequences of SEG-SEM are shown below

SEG (Baba, T. et al., Lancet 359, 1819-1827 (2002)) [SEQ ID NO: 9] 1MNKIFRVLTV SLFFFTFLIK NNLAYADVGV INLRNFYANY QPEKLQGVSS GNFSTSHQLE 61YIDGKYTLYS QFHNEYEAKR LKDHKVDIFG ISYSGLCNTK YMYGGITLAN QNLDKPRNIP 121INLWVNGKQN TISTDKVSTQ KKEVTAQEID IKLRKYLQNE YNIYGFNKTK KGQEYGYKSK 181FNSGFNKGKI TFHLNNEPSF TYDLFYTGTG QAESFLKIYN DNKTIDANF HLDVEISYEK 241 TESEH (Omoe, K. et al., J. Clin. Microbiol. 40: 857-862 (2002)) [SEQ IDNO: 10] 1 EDLHDKSELT DLALANAYGQ YNHPFIKENI KSDEISGEKD LIFRNQGDSGNDLRVKFATA 61 DLAQKFKNKN VDIYGASFYY KCEKISENIS ECLYGGTTLN SEKLAQERVIGANVWVDGIQ 121 KETELIRTNK KNVTLQELDI KIRKILSDKY KIYYKDSEIS KGLIEFDMKTPRDYSFDIYD 181 LKGENDYEID KIYEDNKTLK SDDISHIDVN LYTKKKV SEI (Kuroda, M.et al., Lancet 357 (9264), 1225-1240 (2001)) [SEQ ID NO: 11] 1MKKFKYSFIL VFILLFNIKD LTYAQGDIGV GNLRNFYTKH DYIDLKGVTD KNLPIANQLE 61FSTGTNDLIS ESNNWDEISK FKGKKLDIFG IDYNGPCKSK YMYGGATLSG QYLNSARKIP 121INLWVNGKHK TISTDKIATN KKLVTAQEID VKLRRYLQEE YNIYGHNNTG KGKEYGYKSK 181FYSGFNNGKV LFHLNNEKSF SYDLFYTGDG LPVSFLKIYE DNKIIESEKF HLDVEISYVD 241 SNSEJ (Zhang, S. et al., FEMS Microbial. Lett. 168: 227-233 (1998)) [SEQID NO: 12] 1 MKKTIFILIF SLTLTLLITP LVYSDSKNET IKEKNLHKKS ELSSITLNNLRHIYFFNEKG 61 ISEKIMTEDQ FLDYTLLFKS FFISHSQYND LLVQFDSKET VNKFKGKQVDLYGSYYGFQC 121 SGGKPNKTAC MYGGVTLHEN NQLYDTKKIP INLWIDSIRT VVPLDIVKTNKKKVTIQELD 181 LQARYYLHKQ YNLYNPSTFD GKIQKGLIVF HTSKEPLVSY DLFNVIGQYPDKLLKIYQDN 241 KIIESENMHI DIYLYTSLIV LISLPLVL SEK (Baba, T., et al.,Lancet 359, 1819-1827 (2002)) [SEQ ID NO: 13] 1 MKKLISILLI NIIILGVSNNASAQGDIGID NLRNFYTKKD FINLKDVKDN DTPIANQLQF 61 SNESYDLISE SKDFNKFSNFKGKKLDVFGI SYNGQCNTKY IYGGITATNE YLDKPRNIPI 121 NIWINGNHKT ISTNKVSTNKKFVTAQEIDI KLRRYLQEEY NIYGHNGTKK GEEYGHKSKF 181 YSGFNIGKVT FHLNNNDTFSYDLFYTGDDG LPKSFLKIYE DNKTVESEKF HLDVDISYKE 241 TK SEL (Kuroda, M. etal., Lancet 357, 1225-1240 (2001)) [SEQ ID NO: 14] 1 MKKRLLFVIVITLFIFSSNH TVLSNGDVGP GNLRNFYTKY EYVNLKNVKD KNSPESHRLE 61 YSYKNDTLYAEFDNEYITSD LKGKNVDVFG ISYKYGSNSR TIYGGVTKAE NNKLDSPRII 121 PINLIINGKHQTVTTKSVST DKKMVTAQEI DVKLRKYLQD EFNIYGHNDT GKGKEYGTSS 181 KFYSGFDKGSVVFHMNDGSN FSYDLFYTGY GLPESFLKIY KDNKTVDSTQ FHLDVEISKR SEM (Kuroda, M etal., Lancet 357, 1225-1240 (2001)) [SEQ ID NO: 15] 1 MKRILIIVVLLFCYSQNHIA TADVGVLNLR NYYGSYPIED HQSINPENNH LSHQLVFSMD 61 NSTVTAEFKNVDDVKKFKNH AVDVYGLSYS GYCLKNKYIY GGVTLAGDYL EKSRRIPINL 121 WVNGEHQTISTDKVSTNKKL VTAQEIDTKL RRYLQEEYNI YGFNDTNKGR NYGNKSKFSS 181 GFNAGKILFHLNDGSSFSYD LFDTGTGQAE SFLKIYNDNK TVETEKFHLD VEISYKDES

Streptococcal Pyrogenic Exotoxins (SpEs)

The SpE's SPEA, SPEB, SPEC, SPEG, SPEH, SME-Z, SME-Z₂ and SSA are SAgsinduce tumoricidal effects. SPEA, SPEB, SPEC have been known for sometime and their structures and biological activities described innumerous publications.

SPEG, SPEH, and SPEJ genes were identified from the Streptococcuspyogenes M1 genomic database and described in detail in Proft, T et al.,J. Exp. Med. 189: 89-101 (1999) which also describes SMEZ, SMEZ-2. Thisdocument also describes the cloning and expression of the genes encodingthese proteins.

The smez-2 gene was isolated from the S. pyogenes strain 2035, based onsequence homology to the streptococcal mitogenic exotoxin z (sinez)gene. SMEZ-2, SPE-G, and SPE-J are most closely related to SMEZ andSPEC, whereas SPEH is most similar to the SEs than to any otherstreptococcal toxin.

As described by Proft, T et al supra, rSMEZ, rSMEZ-2, rSPE-G, and rSPE-Hwere mitogenic for human peripheral blood T lymphocytes. SMEZ-2 appearsto be the most potent SAg discovered thus far.

All these toxins, except rSPE-G, were active on murine T cells, but withreduced potency.

Binding to a human B-lymphoblastoid line was shown to be zinc dependentwith high binding affinity of 15-65 nM. Analysis of competition forbinding between toxins of this group revealed overlapping but discretebinding to subsets of class II molecules in the hierarchical order(SMEZ, SPE-C)>SMEZ-2>SPE-H>SPE-G. The most common targets for these SAgswere human Vβ2.1- and Vβ4-expressing T cells.

Streptococcus Pyrogenic Exotoxin A (SPEA)

SPEA can be purified from cultures of S. pyogenes as described by Klineet al., Infect. Immun. 64:861-869 (1996). Plasmids that include thespeaI gene which encode SPEA, and the expression and purification ofrecombinant SPEA (“rSPEA”) are described by Kline et al., supra. Thenative SPEA sequence is shown below:

SPEA (Papageorgiou, A. C. et al. EMBO J. 18: 9-21 (1999)) [SEQ ID NO:16] 1 MENNKKVLKK MVFFVLVTFL GLTISQEVFA QQDPDPSQLH RSSLVKNLQN IYFLYEGDPV61 THENVKSVDQ LLSHDLIYNV SGPNYDKLKT ELKNQEMATL FKDKNVDIYG VEYYHLCYLC 121ENAERSACIY GGVTNHEGNH LEIPKKIVVK VSIDGIQSLS FDIETNKKMV TAQELDYKVR 181KYLTDNKQLY TNGPSKYETG YIKFIPKNKE SFWFDFFPEP EFTQSKYLMI YKDNETLDSN 241TSQIEVYLTT K

Streptococcus Pyrogenic Exotoxin B (SPEB)

Purification of native SpEB is described by Gubba, S. et al., Infect.Immun. 66: 765-770 (1998). Expression and purification of recombinantSpEB are also described in this reference. The native SPEB sequence isshown below (Kapur, V. et al., Microb. Pathog. 15:327-346 (1993)):

[SEQ ID NO: 17] 1 MNKKKLGIRL LSLLALGGFV LANPVFADQN FARNEKEAKD SAITFIQKSAAIKAGARSAE 61 DIKLDKVNLG GELSGSNMYV YNISTGGFVI VSGDKRSPEI LGYSTSGSFDANGKENIASF 121 MESYVEQIKE NKKLDTTYAG TAEIKQPVVK SLLDSKGIHY NQGNPYNLLTPVIEKVKPGE 181 QSFVGQHAAT GCVATATAQI MKYHNYPNKG LKDYTYTLSS NNPYFNHPKNLFAAISTRQY 241 NWNNILPTYS GRESNVQKMA ISELMADVGI SVDMDYGPSS GSAGSSRVQRALKENFGYNQ 301 SVHQINRSDF SKQDWEAQID KELSQNQPVY YQGVGKVGGH AFVIDGADGRNFYHVNWGWG 361 GVSDGFFRLD ALNPSALGTG GGAGGFNGYQ SAVVGIKP

Streptococcus Pyrogenic Exotoxin C(SPEC)

Methods of isolation and characterization of SPEC is carried out by themethods of Li, P L et al., J. Exp. Med. 186: 375-383 (1997). Thesereferences also describe T cell proliferation stimulated by this SAg andthe analysis of its selectivity for TCR Vβ regions. The native sequenceof SPEC (Kapur, V. et al., Infect. Immun. 60:3513-3517 (1992) is shownbelow:)

[SEQ ID NO: 18] 1 MKKINIIKIV FIITVILIST ISPIIKSDSK KDISNVKSDL LYAYTITPYDYKDCRVNFST 61 THTLNIDTQK YRGKDYYISS EMSYEASQKF KRDDHVDVFG LFYILNSHTGEYIYGGITPA 121 QNNKVNHKLL GNLFISGESQ QNLNNKIILE KDIVTFQEID FKIRKYLMDNYKIYDATSPY 181 VSGRIEIGTK DGKHEQIDLF DSPNEGTRSD IFAKYKDNRI INMKNFSHFDIYLE

Streptococcal Superantigen (SSA)

SSA is an ˜28-kDa SAg protein isolated from culture supernatants asdescribed by Mollick J et al., J. Clin. Invest. 92: 710-719 (1993) andReda K et al., Infect. Immun. 62: 1867-1874 (1994). SSA stimulatesproliferation of human T cells bearing Vβ1, Vβ3, Vβ5.2, and Vβ15 in anMHC class II-dependent manner. The first 24 amino acid residues of SSAare be 62.5% identical to SEB, SEC1, and SEC3. Purification and cloningof SSA is described in Reda K et al., Infect. Immun. 62: 1867-1874(1994). The native sequence of SSA (Reda, K. B. et al., Infect. Immun.64: 1161-1165 (1996)) is shown below:

[SEQ ID NO: 19] 1 MNKRIRILVV ACVVFCAQLL SISVFASSQP DPTPEQLNKS SQFTGVMGNLRCLYDNHFVE 61 GTNVRSTGQL LQHDLIFPIK DLKLKNYDSV KTEFNSKDLA AKYKNKDVDIFGSNYYYNCY 121 YSEGNSCKNA KKTCMYGGVT EHHRNQIEGK FPNITVKVYE DNENILSFDITTNKKQVTVQ 181 ELDCKTRKIL VSRKNLYEFN NSPYETGYIK FIESSGDSFW YDMMPAPGAIFDQSKYLMLY 241 NDNKTVSSSA IAIEVHLTKK

Streptococcal Pyrogenic Exotoxins G and H and SMEZ

The sequences of the more recently discovered Streptococcal exotoxinSAgs are provided below:

SPEG (Fraser, J et al., Mol Med Today 6: 125-32 (2000)) [SEQ ID NO: 20]1 DENLKDLKRS LRFAYNITPC DYENVEIAFV TTNSIHINTK QKRSECILYV DSIVSLGITD 61QFIKGDKVDV FGLPYNFSPP YVDNIYGGIV KHSNQGNKSL QFVGILNQDG KETYLPSEVV 121RIKKKQFTLQ EFDFKIRKFL MEKYNIYDSE SRYTSGSLFL ATKDSKHYEV DLFNKDDKLL 181SRDSFFKRYK DNKIFNSEEI SHFDIYLKTY SPEH (Proft, T. et al., J. Exp. Med.189: 89-102 (1999)) [SEQ ID NO: 21] 1 MRYNCRYSHI DKKIYSMIIC LSFLLYSNVVQANSYNTTNR HNLESLYKHD SNLIEADSIK 61 NSPDIVTSHM LKYSVKDKNL SVFFEKDWISQEFKDKEVDI YALSAQEVCE CPGKRYEAFG 121 GITLTNSEKK EIKVPVNVWD KSKQQPPMFITVNKPKVTAQ EVDIKVRKLL IKKYDIYNNR 181 EQKYSKGTVT LDLNSGKDIV FDLYYFGNGDFNSMLKIYSN NERIDSTQFH VDVSIS SMEZ (Proft, T. et al., J. Exp. Med. 191:1765-1776 (2000)) [SEQ ID NO: 22] 1 LEVDNNSLLR NIYSTIVYEY SDTVIDFKTSHNLVTKKLDV RDARDFFINS EMDEYAANDF 61 KAGDKIAVFS VPFDWNYLSK GKVTAYTYGGITPYQKTSIP KNIPVNLWIN RKQIPVPYNQ 121 ISTNKTTVTA QEIDLKVRKF LIAQHQLYSSGSSYKSGKLV FHTNDNSDKY SLDLFYTGYR 181 DKESIFKVYK DNKSFNIDKI GHLDIEIDS

Yersinia pseudotuberculosis Mitogen (Superantigen) (YPM)

Cloning, expression and purification of YPM is described byMiyoshi-Akiyama, T. et al., J. Immunol. 154: 5228-5234 (1995).

The above reference described assays of YPM using lymphoid cells andmurine L cells transfected with human HLA genes, including T cellproliferation and cytokine (IL2) secretion. The sequence of YPM is shownbelow

(Carnoy, C. et al., J. Bacteriol. 184 (16), 4489-4499 (2002)): [SEQ IDNO: 23] 1 MKKKFLSLLT LTFFSGLALA ADYDNTLNSI PSLRIPNIET YTGTIQGKGEVCIRGNKEGK 61 SRGGELYAVL RSTNANADMT LILLCSIRDG WKEVKRSDID RPLRYEDYYTPGALSWIWEI 121 KNNSSEASDY SLSATVHDDK EDSDVLMKCPStaphylococcal Exotoxin like Proteins (SET)

The identification characterization of the SETs (SET-1 and SET-2) andthe cloning and purification of SET-1 is described in Williams, R. J. etal., Infect. Immun. 68: 4407-4414 (2000). This reference discloses thedistribution of the set1 gene among Staphylococcal species and strains.

The set1 nucleotide sequences are deposited in the GenBank databaseunder accession numbers AF094826 (set gene cluster fragment), AF188835(NCTC 6571 sell gene), AF188836 (FR1326 set1gene), and AF188837 (NCTC8325-4 sell gene). Recombinant SET-I protein stimulates production ofthe proinflammatory cytokines IL-1β, IL-6, and TNFα

Preferred Form of Superantigen for Therapeutic Use

A preferred construct for intrathecal and intrapleural use comprises aSAg in native form. In contrast, for systemic use the preferred SAg isone to which humans do not make or make only marginal amountsneutralizing antibody fused recombinantly or biochemically to a highaffinity tumor specific antibody, Fab or single chain Fv. To this end,SAg epitopes in the conjugate which bind endogenous (to includepreexisting) SAg specific antibodies are deleted and/or substituted byalanine or amino acid sequences to which the host does not havepreexisting antibodies. For example, a dominant epitope on SEBrecognized by anti-SEB antibodies is the sequence 225-234 (Nishi et al.,J. Immunol. 158: 247-254 (1997) and an epitope on SEA recognized byanti-SEA antibodies is the sequence 121-149 (Hobieka et al., Biochem.Biophys. Res. Comm. 223: 565-571 (1996). Alternatively, SAgs such as Y.pseudotuberculosis or C. perfringens toxin A or to which humans do nothave preexisting antibodies are used. Y pseudotuberculosis SAg has, inaddition, a natural RGD domain which has tumor-localizing properties.

Functional Homologues and Derivatives of Superantigen Proteins ofPeptides

The present invention contemplates, in addition to native SAgs, the useof homologues of native SAgs that have the requisite biological activityto be useful in accordance with the invention.

Thus, in addition to native SAg protein and nucleic acid compositionsdescribed herein, the present invention encompasses functionalderivatives, among which homologues are preferred. Thus, biologicallyactive homologues of staphylococcal enterotoxins, streptococcalexotoxins. Y. pseudotuberculosis SAg YPM, C. perfringens toxin A, M.arthritides SAgs are included herein.

By “functional derivative” is meant a “fragment,” “variant,” “mutant,”“homologue,” “analogue,” or “chemical derivative. Homologues includefusion proteins, chimeric proteins and conjugates that include a SAgportion fused to or conjugated to a fusion partner polypeptide orpeptide. A functional derivative retains at least a portion of thebiological activity of the native protein which permits its utility inaccordance with the present invention. Such biological activity includesstimulation of T cell proliferation and/or cytokine secretion,stimulation of T cell-mediated cytotoxic activity, as a result ofinteractions of the SAg composition with T cells preferably via the TCRVβ region.

A “fragment” refers to any shorter peptide. A “variant” of refers to amolecule substantially similar to either the entire protein or a peptidefragment thereof. Variant peptides may be conveniently prepared bydirect chemical synthesis of the variant peptide, using methodswell-known in the art.

A homologue refers to a natural protein, encoded by a DNA molecule fromthe same or a different species. Homologues, as used herein, typicallyshare at least about 50% sequence similarity at the DNA level or atleast about 18% sequence similarity at the amino acid level, with anative protein.

An “analogue” refers to a non-natural molecule substantially similar toeither the entire molecule or a fragment thereof.

A “chemical derivative” contains additional chemical moieties notnormally a part of the peptide. Covalent modifications of the peptideare included within the scope of this invention. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the peptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues.

A fusion protein comprises a native SAg, a fragment or a homologue fusedby recombinant means to another polypeptide fusion partner, optionallyincluding a spacer between the two sequences. Preferred fusion partnersare antibodies, Fab fragments, single chain Fv fragments. Other fusionpartners are any peptidic receptor ligand, cytokine, extracellulardomain (“ECD”) of a costimulatory molecule and the like.

The recognition that the biologically active regions of the SEs, forexample, are substantially homologous, i.e., that the sequences aresubstantially similar, enables prediction of the sequences of syntheticpeptides which will exhibit similar biological effects in accordancewith this invention (Johnson, L. P. et al., Mol. Gen. Genet. 203:354-356(1986).

The following terms are used in the disclosure of sequences and sequencerelationships between two or more nucleic acids or polypeptides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or other polynucleotide sequence, or the complete cDNAor polynucleotide sequence. The same is the case for polypeptides andtheir amino acid sequences.

As used herein, “comparison window” includes reference to a contiguousand specified segment of a polynucleotide or amino acid sequence,wherein the sequence may be compared to a reference sequence and whereinthe portion of the sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides or amino acids in length, and optionally can be30, 40, 50, 100, or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the sequence a gap penalty is typically introduced and is subtractedfrom the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well-known in the art. For comparison, optimal alignmentof sequences may be done using any suitable algorithm, of which thefollowing are examples:

-   -   (a) the local homology algorithm (“Best Fit”) of Smith and        Waterman, Adv. Appl. Math. 2: 482 (1981);    -   (b) the homology alignment algorithm (GAP) of Needleman and        Wunsch, J. Mol. Biol. 48: 443 (1970); or    -   (c) a search for similarity method (FASTA and TFASTA) of Pearson        and Lipman, Proc. Natl. Acad. Sci. 85 2444 (1988);

In a preferred method of alignment, Cys residues are aligned.Computerized implementations of these algorithms, include, but are notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG) (Madison,Wis.). The CLUSTAL program is described by Higgins et al., Gene73:237-244 (1988); Higgins et al., CABIOS 5:151-153 (1989); Corpet etal., Nuc Acids Res 16:881-90 (1988); Huang et al., CABIOS 8:155-65(1992), and Pearson et al., Methods in Molecular Biology 24:307-331(1994).

A preferred program for optimal global alignment of multiple sequencesis PileUp (Feng and Doolittle, J Mol Evol 25:351-360 (1987) which issimilar to the method described by Higgins et al. 1989, supra).

The BLAST family of programs which can be used for database similaritysearches includes: NBLAST for nucleotide query sequences againstdatabase nucleotide sequences; XBLAST for nucleotide query sequencesagainst database protein sequences; BLASTP for protein query sequencesagainst database protein sequences; TBLASTN for protein query sequencesagainst database nucleotide sequences; and TBLASTX for nucleotide querysequences against database nucleotide sequences. See, for example,Ausubel et al., eds., Current Protocols in Molecular Biology, Chapter19, Greene Publishing and Wiley-Interscience, New York (1995) or mostrecent edition. Unless otherwise stated, stated sequenceidentity/similarity values provided herein, typically in percentages,are derived using the BLAST 2.0 suite of programs (or updates thereof)using default parameters. Altschul et al., Nuc Acids Res. 25:3389-3402(1997).

As is known in the art, BLAST searches assume that proteins can bemodeled as random sequences. However, many real proteins compriseregions of nonrandom sequence which may include homopolymeric tracts,short-period repeats, or regions rich in particular amino acids.Alignment of such regions of “low-complexity” regions between unrelatedproteins may be performed even though other regions are entirelydissimilar. A number of low-complexity filter programs are known thatreduce such low-complexity alignments. For example, the SEG (Wooten etal., Comput. Chem. 17:149-163 (1993)) and XNU (Claverie et al., Comput.Chem., 17:191-201 (1993)) low-complexity filters can be employed aloneor in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or amino acid sequences refers to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window. It is recognized that when usingpercentages of sequence identity for proteins, a residue position whichis not identical often differs by a conservative amino acidsubstitution, where a substituting residue has similar chemicalproperties (e.g., charge, hydrophobicity, etc.) and therefore does notchange the functional properties of the polypeptide. Where sequencesdiffer in conservative substitutions, the % sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution, and be expressed as “sequence similarity” or “similarity”(combination of identity and differences that are conservativesubstitutions). Means for making this adjustment are well-known in theart. Typically this involves scoring a conservative substitution as apartial rather than as a full mismatch, thereby increasing thepercentage sequence identity. Thus, for example, where an identicalamino acid is given a score of “1” and a non-conservative substitutionis given a score of “0” zero, a conservative substitution is given ascore between 0 and 1. The scoring of conservative substitutions iscalculated, e.g., according to the algorithm of Meyers et al., CABIOS4:11-17 (1988) as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

As used herein, “percentage of sequence identity” refers to a valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the nucleotide or amino acidsequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence (which lacks suchadditions or deletions) for optimal alignment, such as by the GAPalgorithm (supra). The percentage is calculated by determining thenumber of positions at which the identical nucleotide or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing that number by the total number of positions in thewindow of comparison and multiplying the result by 100, therebycalculating the percentage of sequence identity.

The term “substantial identity” of two sequences means that apolynucleotide or polypeptide comprises a sequence that has at least60%, preferably at least 70%, more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 95% sequenceidentity to a reference sequence using one of the alignment programsdescribed herein using standard parameters. Values can be appropriatelyadjusted to determine corresponding identity of the proteins encoded bytwo nucleotide sequences by taking into account codon degeneracy, aminoacid similarity, reading frame positioning, etc.

One indication that two nucleotide sequences are substantially identicalis if they hybridize to one other under stringent conditions. Because ofthe degeneracy of the genetic code, a number of different nucleotidecodons may encode the same amino acid. Hence, two given DNA sequencescould encode the same polypeptide but not hybridize under stringentconditions. Another indication that two nucleic acid sequences aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid. Clearly, then, two peptide orpolypeptide sequences are substantially identical if one isimmunologically reactive with antibodies raised against the other. Afirst peptide is substantially identical to a second peptide, if theydiffer only by a conservative substitution. Peptides which are“substantially similar” share sequences as noted above except thatnonidentical residue positions may differ by conservative substitutions.

Thus, in one embodiment of the present invention, the Lipman-PearsonFASTA or FASTP program packages (Pearson, W. R. et. al., 1988, supra;Lipman, D. J. et al, Science 227:1435-1441 (1985)) in any of its olderor newer iterations may be used to determine sequence identity orhomology of a given protein, preferably using the BLOSUM 50 or PAM 250scoring matrix, gap penalties of ±12 and ±2 and the PIR or SwissPROTdatabases for comparison and analysis purposes. The results areexpressed as z values or E( ) values. To achieve a more “updated” zvalue cutoff for statistical significance, preferably corresponding to az value >10 based on the increase in database size over that of 1988, ina FASTA analysis using the equivalent 2001 database, a significant zvalue would exceed 13.

A more widely used and preferred methodology determines the percentidentity of two amino acid sequences or of two nucleic acid sequencesafter optimal alignment as discussed above, e.g., using BLAST. In apreferred embodiment of this approach, a polypeptide being analyzed forits homology with native SAg is at least 20%, preferably at least 40%,more preferably at least 50%, even more preferably at least 60%, andeven more preferably at least 70%, 80%, or 90% as long as the referencesequence. The amino acid residues (or nucleotides) at correspondingpositions are then compared. Amino acid or nucleic acid “identity” isequivalent to amino acid or nucleic acid “homology”.

In a preferred comparison of a putative SAg homologue polypeptide and anative SAg protein, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch alignmentalgorithm (incorporated into the GAP program in the GCG software package(available at the URL www.gcg.com), using either a Blossom 62 matrix ora PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, thepercent identity between the encoding nucleotide sequences is determinedusing the GAP program in the GCG software package (also available atabove URL), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In anotherembodiment, the algorithm of Meyers et al., supra (incorporated into theALIGN program, version 2.0), is implemented using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

The wild-type (or native) SAg-encoding nucleic acid sequence or the SAgprotein sequence can further be used as a “query sequence” to searchagainst a public database, for example, to identify other family membersor related sequences. Such searches can be performed using the NBLASTand XBLAST programs, supra (see Altschul et al. (1990) J. Mol. Biol.215:403-10). BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to identify nucleotide sequenceshomologous to native SAgs. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to identify amino acidsequences homologous to identify polypeptide molecules homologous to anative SAg. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997, supra).Default parameters of XBLAST and NBLAST can be found at the NCBI website(www.ncbi.nlm.nih.gov)

Using the PASTA programs and method of Pearson and Lipman, a preferredSAg homologue is one that has a z value >10. Expressed in terms ofsequence identity or similarity, a preferred SAg homologue for useaccording the present invention has at least about 20% identity or 25%similarity to a native SAg. Preferred identity or similarity is higher.More preferably, the amino acid sequence of a homologue is substantiallyidentical or substantially similar to a native SAg sequence as thoseterms are defined above.

One group of substitution variants (also homologues) are those in whichat least one amino acid residue in the peptide molecule, and preferably,only one, has been removed and a different residue inserted in itsplace. For a detailed description of protein chemistry and structure,see Schulz, G. E. Principles of Protein Structure Springer-Verlag, NewYork, 1978, and Creighton, T. E., Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, 1983, which are herebyincorporated by reference. The types of substitutions which may be madein the protein or peptide molecule of the present invention may be basedon analysis of the frequencies of amino acid changes between ahomologous protein of different species, such as those presented inTable 1-2 of Schulz et al. (supra) and FIG. 3-9 of Creighton (supra).Based on such an analysis, conservative substitutions are defined hereinas exchanges within one of the following five groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr(Pro, Gly);2. Polar, negatively charged residues and their amides: Asp, Asn, Glu,Gln;3. Polar, positively charged residues: His, kg, Lys;4. Large aliphatic, nonpolar residues: Met, Leu, 11e, Val (Cys); and5. Large aromatic residues: Phe, Tyr, Tip.

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking any side chain andthus imparts flexibility to the chain. Pro, because of its unusualgeometry, tightly constrains the chain. Cys can participate in disulfidebond formation which is important in protein folding. Tyr, because ofits hydrogen bonding potential, has some kinship with Ser, Thr, etc.

More substantial changes in functional or immunological properties aremade by selecting substitutions that are less conservative, such asbetween, rather than within, the above five groups, which will differmore significantly in their effect on maintaining (a) the structure ofthe peptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Examplesof such substitutions are (a) substitution of gly and/or pro by anotheramino acid or deletion or insertion of Gly or Pro; (b) substitution of ahydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobicresidue, e.g., Len, Ile, Phe, Val or Ala; (c) substitution of a Cysresidue for (or by) any other residue; (d) substitution of a residuehaving an electropositive side chain, e.g., Lys, Arg or His, for (or by)a residue having an electronegative charge, e.g., Glu or Asp; or (e)substitution of a residue having a bulky side chain, e.g., Phe, for (orby) a residue not having such a side chain, e.g., Gly.

The deletions and insertions, and substitutions according to the presentinvention are those which do not produce radical changes in thecharacteristics of the protein or peptide molecule. However, when it isdifficult to predict the exact effect of the substitution, deletion, orinsertion in advance of doing so, one skilled in the art will appreciatethat the effect will be evaluated by routine screening assays, forexample direct or competitive immunoassay or biological assay of T cellfunction as described herein. Modifications of such proteins or peptideproperties as redox or thermal stability, hydrophobicity, susceptibilityto proteolytic degradation or the tendency to aggregate with carriers orinto multimers are assessed by methods well known to the ordinarilyskilled artisan.

Chemical Derivatives

Covalent modifications of the SAg proteins or peptide fragments thereof,preferably of SEs or peptide fragments thereof, are included herein.Such modifications may be introduced into the molecule by reactingtargeted amino acid residues of the protein or peptide with an organicderivatizing agent that is capable of reacting with selected side chainsor terminal residues. This may be accomplished before or afterpolymerization.

Cysteinyl residues most commonly are reacted with a-haloacetates (andcorresponding amines), such as 2-chloroacetic acid or chloroacetamide,to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinylresidues also are derivatized by reaction with bromotrifluoroacetone,α-bromo-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing a-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides as noted above. Aspartyl and glutamylresidues are converted to asparaginyl and glutaminyl residues byreaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

Such derivatized moieties may improve the solubility, absorption,biological half life, and the like. The moieties may alternativelyeliminate or attenuate any undesirable side effect of the protein andthe like. Moieties capable of mediating such effects are disclosed, forexample, in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980).

Superantigen Homologues

The variants or homologues of native SAg proteins or peptides includingmutants (substitution, deletion and addition types), fusion proteins (orconjugates) with other polypeptides, are characterized by substantialsequence homology to

(a) the long-known SE's—SEA, SEB, SEC₁₋₃, SED, SEE and TSST-1;(b) long-known SpE's;(c) more recently discovered SE's (SEG, SEH, SEI, SEJ, SEK, SEL, SEM,SETs 1-5); or(d) non-enterotoxin superantigens (YPM, M. arthritides superantigen).Preferred homologues were disclosed above.

Table 1 below lists a number of native SEs and exemplary homologues(amino acid substitution, deletion and addition variants (mutants) andfragments) with z values >10 (range: z=16 to z=136) using theLipman-Pearson algorithm and FASTA. These homologues also inducesignificant T lymphocyte mitogenic responses that are generallycomparable to native SE's.

In addition, as shown in Table 2, several of these homologues alsopromote antigen-nonspecific T lymphocyte killing in vitro by a mechanismtermed “superantigen-dependent cellular cytotoxicity” (SDCC) or, in thecase of SAg-mAb fusion proteins, “superantigen/antibody dependentcellular cytotoxicity (SADCC).

According to the present invention, other SE homologues (e.g., z>10 or,in another embodiment, having at least about 20% sequence identity or atleast about 25% sequence similarity when compared to native SEs),exhibiting T lymphocyte mitogenicity, SDCC or SADCC, are usefulanti-tumor agents when administered to a tumor bearing host via anyintrathecal route.

Tumors in Sheaths Encasing Organs

The appearance of tumors in sheaths (“theca”) encasing an organ oftenresults in production and accumulation of large volumes of fluid in theorgan's sheath. Examples include (1) pleural effusion due to fluid inthe pleural sheath surrounding the lung, (2) ascites originating fromfluid accumulating in the peritoneal membrane and (3) cerebral edema dueto metastatic carcinomatosis of the meninges. Such effusions and fluidaccumulations generally develop at an advanced stage of the disease.

Intrathecal Superantigens for Treatment of Malignant Ascites andMalignant Pleural Effusions

The present invention contemplates the use of any SAg or SET in anyform. This includes but is not limited to staphylococcal enterotoxins A,B, C, D, E, F, G H, I, J, K, L, M, Sp YPM, M. arthritides SAg, C.perfringens exotoxin for direct administration into cavities or spaces,e.g., peritoneum, thecal space, pericardial and pleural space containingtumor.

TABLE 1 SE-Homologues Induce T Lymphocyte Mitogenesis T LymphocyteMitogenic Response ^(b) Reference SE Homologue ^(a) (EC₆₀) ^(c)(SPECIES) SEA (native) 1 Abrahmsen et al., EMBO J. SEA D227A 1057 14:2978-2986 (1995); SEA F47A 52 HUMAN SEA H225A 1272 SEA K123A/D132G 2 SEAN128A 2 SEA K55A 1 SEA H50A 4 SEA D45A 1 SEA H187A 11 SEA E191A/N195A 1SEA C96S 12 Grossman et al., J. SEA C106Q 13 Immunol. 147: 3274-3281 SEAC96, 106G 10 (1991) MOUSE SEA K14E 1 Bavari et al., J. Infect. SEA Y64A100 Dis. 174: 338-345 (1996) SEA Y92A 100 HUMAN SEB (native) 1 Briggs etal., Immunol. SEB H166A/V169E 5 90: 169-175 (1997) SEB H166A 1.3 MOUSESEB V169A 10 SEB V169E 5 SEB V169K 10 SEB (native) 1 Alakhov et al.,Eur. J. SEB (1-13, 2-13) 7.6 Biochem. 209: 823-828 (1992) HUMAN SEB(native) 1 Leder et al., J. Exp. Med. SEB L20T 1.2 187: 823-833 (1998)SEB V26Y 1 MOUSE SEB Y91B 1.8 SEC3 (native) 1 SEC3 Y26A 7 SEC3 N60A 6SEC3 Y90A 8 SEC3 G106A 6 SEC1 (native) 1 Hoffman et al., Infect. SEC1818 (delete 7-9) 1 Immun. 62: 3396-3407 SEC 1819 (delete 6-10) 1 (1994)HUMAN SEC 1820 (delete 9-13) 1 SEC 1821 (delete 9-18) 53 SEC M_(r)(20-80) 4.3 Spero et al., J. Biol. Chem. 24: 8787-8791 (1978) MOUSE SED(native) 1 Sundstrom et al., EMBO J. SED F42A ~100 15: 6832-6840 (1996)SED D182A ~5000 HUMAN SED 218A ~1 SED D222A ~100,000 SEE (native) 1Lamphear et al., J. SEE-Ala (20-24) 1 Immunol. 156: 2178-2185 SEE-Ala(200-207 1 (1996) HUMAN SEE-Ala (20-24/200-207) 1.7 SEA (native) 1Mollick et at., J. Exp. Med. SEA-SEE (200-207) 1 283-293 (1993) HUMANSEE-SEA (70-71) SEA-SEE (200-207) 1 TSST-1 (native) 1 Kum et al., J.Infect. Dis. G31R 800 174: 1261-1270 (1996) HUMAN SEA-C215 mAb Fab 1Antonnson et al., J. Fusion Protein Immunol. 158: 4245-4251 SEE-C215 mAbFab 10 (1997) HUMAN Fusion protein SEE/AA-C215 mAb Fab 1 Fusion proteinSEE/A-C-C215 mAb Fab 10 Fusion protein SEE/A-F-C215 mAb Fab 10 Fusionprotein SEE/A-H-C215 mAb Fab 10 Fusion protein SEA/E-BDEG-mAb Fab 2Fusion protein SEE/A-AH-215mAb Fab 2 Fusion protein LEGEND FOR TABLE 1(above) ^(a) z values for homologues range from 16-136. ^(b) Summary ofMethods in all the above studies: human peripheral blood mononuclearcells (PBMC) or mouse spleen or lymph node lymphocytes were incubatedwith native SE or homologue (mutant) in complete medium supplementedwith fetal calf serum (5 or 10% v/v) and antibiotics in wells of 96-wellmicroplates in 200 μl volumes. In some cases, enriched or purified Tlymphocytes from these populations were tested. Between 0.2 × 10⁵ and 8× 10⁵ cells/well were used. Incubation was at 37° C. in humidifiedair/95% CO₂ for periods of between 66 hours and 84 hours (depending onwhether unfractionated or purified T lymphocytes were being used). Tlymphocyte mitogenic responses was routinely measured as radiolabeled[³H]-thymidine (“TdR”) incorporation during the final 4-24 hrs ofincubation. Cells were always harvested from the microplates onto glassfiber filters which were dried and placed in a liquid scintillationcounter for evaluation of incorporated radio label. ^(c) Each SE orhomologue was tested over a range of concentrations and the results wereplotted as counts/min (cpm) of [³H]TdR taken up (after subtraction ofbackground cpm of cells incubated in medium alone, which rarely exceededseveral hundred cpm) on the ordinate vs. log concentration of the SE orhomologue on the abscissa. For each agent tested, the concentration atwhich [³H]TdR incorporation was 50% of maximum (the EC₅₀), which fallsin the linear part of the sigmoid dose-response curve, has been providedin the publication or interpolated visually and approximated (valuepreceded by “~” symbol) from the published graphs. The EC₅₀ of thenative SE was arbitrarily set to 1, so an EC₅₀ of 10 for a homologueindicates that the homologue causes half-maximal mitogenicresponsiveness at a 10-fold higher concentration.

TABLE 2 SE Homologues Induce T Lymphocyte Mitogenesis and Anti-TumorEffects In Vitro T Lymphocyte Mitogenic Response¹ SDCC² SADCC³ SEHomologue (EC₅₀) (EC₅₀) (% of native SE) Data from: Abrahmsen et al.,EMBO J. 14: 2978-2986 (1995) WO96/01650 SEA (native) 1 1 100 SEA D227A1057 132 100 SEA F47A 52 4 100 SEA H225A 1272 130 nd SEA K123A/D132G 2 2100 SEA N128A 2 3 100 SEA K55A 1 1 nd SEA H50A 4 2 100 SEA D45A 1 1 ndSEA H187A 11 9 100 SEA E191A/N195A 1 1 nd Data from Sundstrom et at.,EMBO J. 15:6832-6840 (1996) SED (native) 1 1 SED F42A ~100 ~5 SED D182A~5000 ~50 SED H218A ~1 ~1 SED D222A ~50,000 ~50 Data from Nilsson etal., J. Immunol. 163: 6686-6693 (1999) SEH (native) 1 1 SEH D167 10 5SEN D203A 7 5 SHE D208A 300 10 LEGEND FOR TABLE 2 (above): ¹Lymphocyteproliferation assays: (a) Abrahmsen et al., 1995: Peripheral bloodmononuclear cells (PBMC) from heparinized blood of normal donors wereisolated by density centrifugation over Ficoll-Hypaque. Following this,2 33 10⁵ PBMC/0.2 ml complete medium were incubated in microplates withvarying amounts of SEA or SEA mutants for 72 h and tested for mitogenicresponses (proliferation) by incorporation of [³H]-thymidine during thelast 4 h of culture. The SEA mutant concentration resulting inhalf-maximum proliferation (EC₅₀) was related to the EC₅₀ of the nativeSE, arbitrarily set to 1 (see column 2). Thus, the SEA homologueconcentration to induce half maximal response was related to the samevalues induced by native SEA. (b) Sundstrom et. at., 1996: 10⁵ humanPBMC prepared as above were incubated at 37°0 C. in 0.2 ml completemedium in U-shaped microplate wells with varying amounts of native SEDor SED mutants for 96 hrs. Proliferation was estimated by incorporationof [³H]thymidine added during the final 24 hrs.EC_(50 values were estimated by interpolating the curves in this publication.)(c) Nilsson et al., 1999: 2 × 10⁵ human PBMC were prepared as aboveincubated in flat bottom microwells in 0.2 ml volumes at 37° C. for 72 hwith varying amounts of native SEH and variants. Each well was pulsedwith 0.5 μCi [³H] thymidine for 4 h. Cells were harvested andproliferation measured as incorporation of [3H] thymidine. The EC₅₀values of the SEH variants were related to the EC₅₀ of native SEH whichwas 0.2 pM. ²SDCC = Superantigen dependent mediated cellularcytotoxicity. This assay measures the ability of an SE (whether nativeor mutant) to target cytotoxic T lymphocytes onto MHC class II+ targetcells resulting in their lysis. The same conditions were used in theabove publications. The cytotoxicity of SE (wt) and homologues againstMHC class II+ Raji cells was analyzed in a standard 4 or 6 hour ⁵¹Cr-release assay, using SE-specific T cell lines that had beenstimulated in vitro (with the wild-type SE) as effector cells. Briefly,2.5 × 10³ ⁵¹ Cr-labeled Raji cells were incubated in 0.2 ml medium(RPMI, 10% FCS) in microwells in the presence effector cells at aneffector:target cell ratio of 30 and in the presence (or absence fornegative controls) of the SE's or homologues. After incubation, 0.1 mlof medium was withdrawn and counted in a gamma counter to determineisotope release. % specific cytotoxicity was calculated as$100 \times {\frac{\left( {{{c.p.m}\mspace{14mu} {experimental}\mspace{14mu} {release}} - {{c.p.m}\mspace{14mu} {background}\mspace{14mu} {release}}} \right)}{\left( {{{c.p.m.\mspace{14mu} {total}}\mspace{14mu} {release}} - {{c.p.m}\mspace{14mu} {background}\mspace{14mu} {release}}} \right)}.}$The SE homologue concentration resulting in half-maximum cytotoxicity(EC₅₀) was related to the EC₅₀ of the native SE, arbitrarily set to 1.Thus, the SE homologue concentration needed to promote half maximalcytotoxicity was related to the same values induced by the native orwild SE. EC₅₀ values were provided by the authors, or,in the case of theLundstrom reference, they were estimated by interpolating the curves inthis publication (shown as approximate using the ~ symbol. ³SADCC =Superantigen-tumor specific antibody mediated cellular cytotoxicity.This is similar to SDCC but involves an antibody component in the formof a fusion protein that directs the specificity of the targeting. Here,this assay measure the ability of a fusion protein comprising an SE(native or mutant) fused to an antibody Fab fragment to targetactivatedcytotoxic T lymphocytes onto tumor cells expressing the tumor antigen(colon cancer antigen) against which the antibody (C215) is specific.This targeting leads to tumor cell lysis, as above. The cytotoxicity ofC215Fab-SEA(wt), C215Fab-SEA(m), SEA(wt) and SEA mutants against C215 +MHC class II^(neg) colon carcinoma cells SW 620 was analyzed inastandard 4 hour ⁵¹Cr³⁺⁻ release assay, using in vitro stimulated SEAspecific T cell lines as effector cells. Briefly, ⁵¹Cr³⁺-labeled SW 620cells were incubated at 2.5 × 10³ cells per 0.2 ml medium {RPMI, 10%FCS) in microtiter wells at effector to target cell ratio 30:1 inthepresence or absence (control) of the additives. Percent specificcytotoxicity was calculated as for SDCC assays.

The present invention contemplates the direct administration of any SAg(SEA, SEB and SEC are preferred) into a fluid space containing tumorcells or adjacent to membranes such as pleural, peritoneal, pericardialand thecal spaces containing tumor. These sites display malignantascites, pleural and pericardial effusions or meningeal carcinomatosis.The SAg is preferably administered after partial or complete drainage ofthe fluid (e.g., ascites, pleural or pericardial effusion) but it mayalso be administered directly into the undrained space containing theeffusion, ascites and/or carcinomatosus. In general, the SE dose mayvary from 1 picogram to 10 μg and given every 3 to 10 clays. It iscontinued until there is no reaccumulation of the ascites or effusion.Therapeutic responses are considered to be no further accumulation offour weeks after the last intrapleural administration. See Example 1 forfurther description of treatment.

Fusion Partners for Native SEs or SE Homologues

Antibodies

Fusion protein partners for the SAg include tumor specific antibodies,preferably F(ab′)₂, Fv or Fd fragments thereof, that are specific forantigens expressed on the tumor. In another embodiment, a fusion partneris a polypeptide ligand f for a receptor expressed on tumor cells. Theseantibodies, fragments or receptor ligands may be in the form ofsynthetic polypeptides. The nucleic acid form of the antibody isenvisioned which is useful as a fusion construct with the SAg DNA.

One advantage of certain antibody constructs of the present fusionpolypeptides is prolonged half-life and enhanced tissue penetration.Intact antibodies in which the Fc fragment of the Ig chain is presentwill exhibit slower blood clearance than their Fab' fragmentcounterparts, but a fragment-based fusion polypeptide will generallyexhibit better tissue penetrating capability.

Preferentially, the tumor targeting structure in the SAg conjugate(e.g., tumor specific antibody, Fab or single chain Fv fragments ortumor receptor ligand) has a greater affinity for the tumor than the SAgin the conjugate has for the class II molecule thus preventing the SAgfrom binding all MHC class II receptors and favoring binding of theconjugate to the tumor. In the case of SEB, the dominant epitope forneutralizing antibodies 225-234 is recombinantly or biochemically boundto the tumor targeting molecule e.g., tumor specific antibodies, Fas orFv fragments. In so doing, it sterically interferes with the recognitionof the dominant epitope by preexisting antibodies.

To further enhance the affinity of the tumor specific antibody in theconjugate for tumor cells in vivo, tumor specific antibodies are usedwhich are specific for more than one antigenic structures on the tumor,tumor stroma or tumor vasculature or any combination thereof. The tumorspecific antibody or F(ab′)₂, Fab or single chain Fv fragments are monoor divalent like IgG, polyvalent for maximal affinity like IgM orchimeric with multiple tumor (tumor stroma or tumor vasculature)specificities. Thus, when the SE or SPE-MoAb conjugate is administeredin vivo, it will preferentially bind to tumor cells rather than toendogenous SE antibodies or MHC class II receptors.

To reduce affinity of the SE-mAb conjugate for endogenous MHC class IIbinding sites, the high affinity Zn⁺⁺ dependent MHC class II bindingsites in SEA, SEC2, SEC3, SED, SPEA, SPEC, SPEG, SPEH, SMEZ, SMEZ2, M.arthritides are deleted or replaced by inert sequence(s) or aminoacid(s). These structural alterations in SE or SPEA reduce the affinityfor MHC class II receptors from a K_(d) of 10⁻⁷ or 10⁻⁸ to 10⁻⁵. SEB,SEC and SSA and other SEs or SPEs do not have a high affinity Zn++dependent MHC class II binding site but have multiple low affinity MHCclass II binding sites (K_(d) 10⁻⁵-10⁻⁷). In these cases, alteration ofthe MHC class II binding sites is not always necessary to further reduceaffinity for MHC class II receptors; at the very least mutation of oneor two of the low affinity MHC class II binding sites will suffice inmost instances.

Most importantly, tumor specific antibodies, Fab, F(ab′)₂ or singlechain Fab or Fv fragments in the SAg-mAb conjugate have a higheraffinity for tumor antigens (K_(d) 10⁻¹¹-10⁻¹⁴ or lower) than for theSAg has for MHC class II binding sites (K_(d) 10⁻⁵ to 10⁻⁷) and itsdominant epitope has for SAg specific antibodies (K_(d) 10⁻⁷ to 10⁻¹¹).In this way, the conjugate will bind preferentially to the tumor targetin vivo rather than preexisting antibodies or MHC class II receptors.

Antibody fragments are obtained using conventional proteolytic methods.Thus, a preferred procedure for preparation of F(ab′)₂ fragments fromIgG of rabbit and human origin is limited proteolysis by the enzymepepsin. Rates of digestion of an IgG molecule may vary according toisotype; conditions are chosen to avoid significant amounts ofcompletely degraded IgG as is known in the art.

Fab fragments include the constant domain of the light chain (C_(L)) andthe first constant domain (C_(H1)) of the heavy chain. Fab' fragmentsdiffer from Fab fragments by the addition of a few residues at theC-terminus of C_(H1) domain including one or more cysteine(s) from theantibody hinge region. F(ab′)₂ fragments were originally produced aspairs of Fab' fragments that have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,con-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains that enables thescFv to form the desired structure for antigen binding.

The following documents, incorporated by reference, describe thepreparation and use of functional, antigen-binding regions ofantibodies: U.S. Pat. Nos. 5,855,866; 5,965,132; 6,051,230; 6,004,555;and 5,877,289.

“Diabodies” are small antibody fragments with two antigen-binding sites,which fragments comprise a heavy chain variable domain (V_(H)) connectedto a light chain variable domain (V_(L)) in the same polypeptide chain(V_(H) and V_(L)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described in EP 404,097 and WO93/11161, incorporated herein by reference. “Linear antibodies”, whichcan be bispecific or monospecific, comprise a pair of tandem Fd segments(V_(H)-C_(H1)-V_(H)-C_(H1)) that form a pair of antigen binding regions.

An antibody fragment may be further modified to increase its half-lifeby any of a number of known techniques. Conjugation to non-proteinpolymers, such PEG and the like, is also contemplated

The antibody fusion partner for use in the present invention may bespecific for tumor cells, tumor stroma or tumor vasculature. Antigensexpressed on tumor cells that are suitable targets for mAb-SAg fusionprotein therapy include erb/neu, MUC1, 5T4 and many others. Antibodiesspecific for tumor vasculature bind to a molecule expressed or localizedor accessible at the cell surface of blood vessels, preferably theintratumoral blood vessels, of a vascularized tumor. Such moleculesinclude endoglin (TEC-4 and TEC-11 antibodies), a TGFβ. receptor,E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a VEGF/VPF receptor, anFGF receptor, a TIE, an α_(ν)β₃ integrin, pleiotropin, endosialin andMHC class II proteins. Such antibodies may also bind tocytokine-inducible or coagulant-inducible products of intratumoral bloodvessels. Certain preferred agents will bind to aminophospholipids, suchas phosphatidylserine or phosphatidylethanolamine

A tumor cell-targeting antibody, or an antigen-binding fragment thereof,may bind to an intracellular component that is released from a necroticor dying tumor cell. Preferably such antibodies are mAbs or fragmentsthereof that bind to insoluble intracellular antigen(s) present in cellsthat may be induced to be permeable, or in cell ghosts of substantiallyall neoplastic and normal cells, but are not present or accessible onthe exterior of normal living cells of a mammal.

Anti-tumor stroma antibodies bind to a connective tissue component, abasement membrane component or an activated platelet component; asexemplified by binding to fibrin, RIBS (receptor-induced binding site)or LIBS (ligand-induced binding site).

Fusion protein optionally include linkers or spacers. Numerous types ofdisulfide-bond containing linkers are known that can be successfullyemployed to fuse the SAg to an antibody or fragment, certain linkers arepreferred based on differing pharmacological characteristics andcapabilities. For example, linkers that contain a disulfide bond that issterically “hindered” are preferred, due to their greater stability invivo, thus preventing release of the SAg moiety prior to binding at thesite of action.

Coaguligand

SAgs may be conjugated to, or operatively associated with, polypeptidesthat are capable of directly or indirectly stimulating coagulation, thusforming a “coaguligand” (Barinaga M et al., Science 275:482-4 (1997);Huang X et al., Science 275:547-50 (1997); Ran S et al., Cancer Res 1998Oct. 15; 58(20):4646-53; Gottstein C et al., Biotechniques 30:190-4(2001)).

In coaguligands, the antibody may be directly linked to a direct orindirect coagulation factor, or may be linked to a second binding regionthat binds and then releases a direct or indirect coagulation factor.The ‘second binding region’ approach generally uses a coagulant-bindingantibody as a second binding region, thus resulting in a bispecificantibody construct. The preparation and use of bispecific antibodies ingeneral is well known in the art, and is further disclosed herein.

Coaguligands are prepared by recombinant expression. The nucleic acidsequences encoding the SAg are linked, in-frame, to nucleic acidsequences encoding the chosen coagulant, to create an expression unit orvector. Recombinant expression results in translation of the new nucleicacid, to yield the desired protein product.

Where coagulation factors are used in connection with the presentinvention, any covalent linkage to the SAg should be made at a sitedistinct from the functional coagulating site. The compositions are thus“linked” in any operative manner that allows each region to perform itsintended function without significant impairment. Thus, the SAg binds toand stimulates T cells, and the coagulation factor promotes bloodclotting.

Preferred coagulation factors are Tissue Factor (“TF”) compositions,such as truncated TF (“tTF”), dimeric, multimeric and mutant TFmolecules. tTF is a truncated TF that is deficient in membrane bindingdue to removal of sufficient amino acids to result in this loss.“Sufficient” in this context refers to a number of transmembrane aminoacids originally sufficient to insert the TF molecule into a cellmembrane, or otherwise mediate functional membrane binding of the TFprotein. The removal of a “sufficient amount of transmembrane spanningsequence” therefore creates a tTF protein or polypeptide deficient inphospholipid membrane binding capacity, such that the protein issubstantially soluble and does not significantly bind to phospholipidmembranes. tTF thus substantially fails to convert Factor VII to FactorVIIa in a standard TF assay yet retains so-called catalytic activityincluding the ability to activate Factor X in the presence of FactorVIIa.

U.S. Pat. No. 5,504,067, specifically incorporated herein by reference,describes tTF proteins. Preferably, the TFs for use herein willgenerally lack the transmembrane and cytosolic regions (amino acids220-263) of the protein. However, the tTF molecules are not limited tothose having exactly 219 amino acids.

Any of the truncated, mutated or other TF constructs may be prepared indimeric form employing the standard techniques of molecular biology andrecombinant expression, in which two coding regions are arrangedin-frame and are expressed from an expression vector. Various chemicalconjugation technologies may be employed to prepare IF dimers.Individual TF monomers may be derivatized prior to conjugation.

The tTF constructs may be multimeric or polymeric, which means that theyinclude 3 or more TF monomeric units. A “multimeric or polymeric TFconstruct” is a construct that comprises a first monomeric TF molecule(or derivative) linked to at least a second and a third monomeric TFmolecule (or derivative). The multimers preferably comprise betweenabout 3 and about 20 such monomer units. The constructs may be readilymade using either recombinant techniques or conventional syntheticchemistry.

TF mutants deficient in the ability to activate Factor VII are alsouseful. Such “Factor VII activation mutants” are generally definedherein as TF mutants that bind functional Factor VII/VIIa,proteolytically activate Factor X, but substantially lack the ability toproteolytically activate Factor VII.

The ability of such Factor VII activation mutants to function inpromoting tumor-specific coagulation is requires their delivery to thetumor vasculature and the presence of Factor VIIa at low levels inplasma. Upon administration of a conjugate of a Factor VII activationmutant, the mutant will be localize within the vasculature of avascularized tumor. Prior to localization, the TF mutant would begenerally unable to promote coagulation in any other body sites, on thebasis of its inability to convert Factor VII to Factor VIIa. However,upon localization and accumulation within the tumor region, the mutantwill then encounter sufficient Factor VIIa from the plasma in order toinitiate the extrinsic coagulation pathway, leading to tumor-specificthrombosis. Exogenous Factor VIIa could also be administered to thepatient to interact with the TF mutant and tumor vasculature.

Any one or more of a variety of Factor VII activation mutants may beprepared and used in connection with the present invention. The FactorVII activation region generally lies between about amino acid 157 andabout amino acid 167 of the TF molecule. Residues outside this regionmay also prove to be relevant to the Factor VII activating activity.Mutations are inserted into any one or more of the residues generallylocated between about amino acid 106 and about amino acid 209 of the TFsequence (WO 94/07515; WO 94/28017; each incorporated herein byreference).

A variety of other coagulation factors may be used in connection withthe present invention, as exemplified by: the agents set forth below.Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIa andderivatives, Factor IX/IXa and derivatives, Factor X/Xa and derivatives,Factor XI/XIa and derivatives, Factor XII/XIIa and derivatives, FactorXIII/XIIIa and derivatives, Factor X activator and Factor V activatormay be used in the present invention. These conjugates are administeredintrathecally in dosages of 0.01 ng/kg to 100 μg/kg.

Cytokines as Fusion Partners

Cytokines are an effective partner for SAgs. Various cytokines, such asIL-2, IL-3, IL-7, IL-12, and IL-18, may be used.

A preferred fusion polypeptide comprises a SAg fused to anti-apoptoticcytokines. SAg stimulation of T cells can result in activation-drivencell death. Several cytokines and bacterial lipopolysaccharide (LPS) areknown to interfere with this process (Vella et al., Proc. Natl. Acad.Sci. 95: 3810-3815 (1998)). IL-3, IL-7, IL-15 and IL-17 preventSAg-stimulated T cells from undergoing apoptosis in vivo and in vitro.In addition, because of their ability to promote selective proliferationby Th₁ T cells, IL-12 and IL-18 are desirable. IL-18 is preferred forintratumoral injection because it induces tumor suppressive cytokinesIFNγ and TNFα and IL-1β, and rescues cytotoxic T cells from apoptosis.

Accordingly, SAg-mAb conjugate as described above is fused recombinantlyto the extracellular domains of at least one cytokine from a groupconsisting of IL-2. IL-7 or IL-3 or IL-12 or IL-15 or IL-17 or IL-18.Other anti-T cell apoptosis agents such as LPS preparations of lowvirulence or a lipid A component (modified to induce less toxicity) arealso effective antiapoptotic agents when conjugated biochemically to theSAg-MoAb (or F(ab′)₂, Fab, Fd or single chain Fv fragments) conjugate orif administered concomitantly with the SAg. Nucleic acids encoding thecytokine of choice is fused in frame with nucleic acids encoding theSAg. These conjugates are administered parenterally, intrathecallyand/or intratumorally by infusion or injection in dosages of 0.01 ng/kgto 100 μg/kg.

Costimulatory Molecules as Fusion Partners

Superantigens Conjugated to OX40L or 4-1BBL

A preferred fusion polypeptide comprises a SAg fused recombinantly to apotent costimulatory molecule, preferably the ECD of a transmembranecostimulatory protein. Examples of such costimulatory molecules are theOX-40 ligand (Godfrey et al., J. Exp. Med. 180: 757-762 (1994);Gramaglia I et al., J. Immunol. 161: 6510-6517 (1998); Maxwell J R etal., J. Immunol. 164: 107-112 (2000) or 4-1BB ligand (Kown B S et al.,Proc. Natl. Acad. Sci. USA 86:1963-67 (1989); Shuford W W et al., J.Exp. Med. 186: 47-55 (1997) and CD-38 (Jackson DG et al., J. Immunol.144: 2811-2817 (1990); Zilber et al., Proc. Nat'l Acad. Sci. USA 97:2840-2845 (2000). The preparation of such fusion proteins is achieved byrecombinant methods in which nucleic acids encoding SAgs are fused inframe to nucleic acids encoding the ECD of the costimulatory moleculesuch as OX-40L (Godfrey et al., J. Exp. Med. 180:757-762 (1994)) or4-1BBL (Goodwin et al. Eur. J. Immunol. 23: 2631-2641 (1993); Melero I.et al., Eur. J. Immunol. 28: 1116-1121 (1998)).

It is preferred to delete from the conjugates or fusion polypeptides ofthe present invention any SAg epitope that binds to SAg-specificantibodies, including preexisting or natural antibodies). Such epitopesare deleted or substituted by Ala or by amino acid sequences notrecognized by preexisting host antibodies. For example, a dominantepitope of SEB that is recognized by anti-SEB antibodies is the sequenceat residues 225-234 (Nishi et al., J. Immunol. 158: 247-254 (1997). Anepitope of SEA that is recognized by anti-SEA antibodies is the sequenceat residues 121-149 (Hobieka et al., Biochem. Biophys. Res. Comm. 223:565-571 (1996). Alternatively, to avoid issues with such preexistingimmunity, SAgs such as YPM or C. perfringens toxin A to which humans donot have preexisting antibodies are selected. YPM, in addition, anatural RGD domain which gives it tumor localizing properties. The SEmay be modified to reduce toxicity by altering its MHC class II bindingaffinity (e.g., SEA D227A-high affinity Zn++ dependent binding site).

Preferably, the tumor targeting structure in SAg conjugate (e.g., tumorspecific antibody or fragment, or a tumor receptor ligand) has greateraffinity for the tumor than the affinity of the SAg in the conjugate forthe MHC class II molecule thus preventing the SAg from binding“promiscuously” to all MHC class II molecules receptors and favoringbinding to the tumor. In the case of SEB, the dominant epitope forneutralizing antibodies, residues 225-234, is recombinantly orbiochemically conjugated to the tumor targeting molecule (e.g., tumorspecific antibody, etc.) so that it can sterically interfere with therecognition of the dominant epitope by preexisting antibodies in thehost.

To further enhance the affinity of the tumor specific antibody in thefusion polypeptide for tumor cells in vivo, one preferably selects atumor specific antibody that is specific for more than one antigenicstructures of the tumor, the tumor stroma or the tumor vasculature (orany combination). The tumor specific antibody or antigen-bindingfragment thereof can be made mono or divalent (like IgG), polyvalentlike IgM to increase avidity or chimeric with multiple tumorspecificities as described above. Thus, when the SAg-mAb conjugate isadministered in vivo, it will preferentially bind to tumor cells ratherthan to endogenous anti-SAg antibodies or MEC class II receptors.

To reduce affinity of the SAg-mAb conjugate for endogenous MHC class IIbinding sites, the high affinity Zn⁺⁺ MHC class II binding site presentin a number of SAgs (SEA, SEC2, SECS, SED, SPEA, SPEC, SPEG, SPEH, SMEZ,SMEZ2, M. arthritides SAg) is deleted or replaced by an “inert”sequence(s) or amino acid. Such structural alterations in SE or SPEA areknown to reduce the affinity for MHC class II from a K_(d) of 10⁻⁷ or10⁻⁸ to a K_(d) of 10⁻⁵. SEB, SEC and SSA and other SAgs do not havesuch a high affinity Zn⁺⁺-dependent MHC class II binding site but havemultiple low affinity MHC class II binding sites (K_(d) of 10⁻⁵-10⁻⁷).In these cases, alteration of the MEC class II binding sites is notalways necessary to further reduce affinity for MHC class II; mutationof one or two of the low affinity MHC class II binding sites willsuffice in most instances.

Most importantly, tumor specific antibodies or their fragments in aSAg-mAb conjugate have higher affinities for tumor antigens (K_(d) of10⁻¹¹-10⁴⁴ or lower) than (a) the affinity of the SAg for MHC class IIbinding sites (K_(d) 10⁻⁵ to 10⁻⁷) or (b) the affinity a dominant SAgepitope for a SAg-specific antibody (K_(d) 10⁻⁷ to 10⁻¹¹). Because ofthis, the conjugate will bind preferentially to the tumor target in vivo

SAg-OX-40 ligand (OX-40L) or 4-1BB ligand (4-1BBL) are fused to a tumorspecific targeting structure using recombinant SAgs. A most preferredconstruct combines the ECD of OX-40L or 4-1BBL with a high affinitytumor specific Fv antibody fragments. The nucleic acids encoding the ECDof OX-40L (Godfrey et al., supra or 4-1BBL (Goodwin et al., Eur. J.Immunol. 23: 2631-2641 (1993); Melero I. et al., Eur. J. Immunol. 28:1116-1121 (1998)) are fused in frame with nucleic acids encoding a SAgof any type, although SEA, SEB, SEC and Y. pseudotuberculosis arepreferred. The SAg may be modified to reduce antigenicity by modifying adominant epitope and to reduce toxicity by altering its MHC class IIbinding affinity as described above. The tumor targeting structure mayinclude but is not limited to a tumor receptor ligand or tumor-specificantibody or a fragment thereof. Preferably, the affinity of the tumortargeting structure is of higher affinity than is the affinity of themodified SAg for MEC class II. High affinity scFv constructs specificfor the OX-40 receptor and 4-1BB receptor may be used in place of theOX40L and 4-1BBL in the SAg-tumor targeting construct.

The SE-OX-40L (or 4-1BB) conjugates described above are administeredparenterally, intratumorally, intrathecally (e.g., intraperitoneally,intrapleurally) by infusion or injection in conventional or sustainedrelease vehicles in dosages of 0.01 ng/kg to 100 μg/kg using standardprotocols or those exemplified herein. Frequency of administration maybe every 3-7 days.

Biochemical Cross-Linkers

In the above fusion polypeptides or conjugates, the SAgs may be linkeddirectly to a fusion partner or fused/conjugated via certain preferredbiochemical linker or spacer groups. For chemical conjugates,cross-linking reagents are preferred and are used to form molecularbridges that bond together functional groups of two different molecules.Heterobifunctional crosslinkers can be used to link two differentproteins in a step-wise manner while preventing unwanted homopolymerformation. Such cross-linkers are listed in Table 3, below.

Hetero-bifunctional cross-linkers contain two reactive groups one (e.g.,N-hydroxy succinimide) generally reacting with primary amine group andthe other (e.g., pyridyl disulfide, maleimides, halogens, etc.) reactingwith a thiol group. Compositions to be crosslinked therefore generallyhave, or are derivatized to have, a functional group available. Thisrequirement is not considered to be limiting in that a wide variety ofgroups can be used in this manner. For example, primary or secondaryamine groups, hydrazide or hydrazine groups, carboxyl, hydroxyl,phosphate, or alkylating groups may be used for binding orcross-linking_(—)

The spacer arm between the two reactive groups of a cross-linker may beof various length and chemical composition. A longer, aliphatic spacerarm allows a more flexible linkage while certain chemical groups (e.g.,benzene group) lend extra stability or rigidity to the reactive groupsor increased resistance of the chemical link to the action of variousagents (e.g., disulfide bond resistant to reducing agents). Peptidespacers, such as Leu-Ala-Leu-Ala, are also contemplated.

It is preferred that a cross-linker have reasonable stability in blood.Numerous known disulfide bond-containing linkers can be used toconjugate two polypeptides. Linkers that contain a disulfide bond thatis sterically hindered may give greater stability ha vivo, preventingrelease of the agent prior to binding at the desired site of action.

A most preferred cross-linking reagents for use in with antibody chainsis SMPT, a bifunctional cross-linker containing a disulfide bond that is“sterically hindered” by an adjacent benzene ring and methyl groups.Such steric hindrance of the disulfide bond may protect the bond fromattack by thiolate anions (e.g., glutathione) which can be present intissues and blood, and thereby help in preventing decoupling of theconjugate prior to the delivery to the target, preferably tumor, site.SMPT cross-links functional groups such as —SH or primary amines (e.g.,the s-amino group of Lys).

TABLE 3 Hetero-Bifunctional Cross-linkers Spacer arm length LinkerAdvantages and Applications after cross linkingSuccinimidyloxycarbonyl-α-(2- Greater stability 11.2 Apyridyldithio)toluene (SMPT) ¹ N-succinimidyl 3-(2- Thiolation  6.8 Apyridyldithio)propionate (SPDP) ² Sulfosuccinimidyl-6-[α-methyl-α-(2-Extended spacer arm; Water-soluble 15.6 Apyridyldithio)toluamido]hexanoate (Sulfo-LC-SPDP) ¹ Succinimidyl-4-(N-Stable maleimide reactive group; 11.6 A maleimidomethyl)cyclohexane-1-conjugation of enzyme or other carboxylate (SMCC) ¹ polypeptide toantibody Succimimidyl-4-(N- Stable maleimide reactive group; 11.6 Amaleimidomethyl)cyclohexane- water-soluble carboxylate (Sulfo-SMCC) ¹m-Maleimidobenzoyl-N- Enzyme-antibody conjugation;  9.9 Ahydroxysuccinimide (MBS) ¹ hapten-carrier protein conjugationm-Maleimidobenzoyl-N- Water-soluble  9.9 A hydroxysulfosuccinimide(Sulfo-MBS) ¹ N-Succinimidyl(4- Enzyme-antibody conjugation 10.6 Aiodacetyl)aminobenzoate (SIAB) ¹ Sulfosuccinimidyl(4- Water-soluble 10.6A iodoacetyl)aminobenzoate (Sulfo- SIAB) ¹ Succinimidyl-4-(p-Enzyme-antibody conjugation; 14.5 A maleimidophenyl)butyrate (SMPB) ¹extended spacer arm Sulfosuccsnimidyl-4-(p- Extended spacer arm 14.5 Amaleimidophenyl)butyrate (Sulfo- Water-soluble SMPB) ¹1-ethyl-3-(3-dimethylaminopropyl) Hapten-Carrier conjugation 0carbodiimide hydrochloride (EDC) + N-hydroxysulfosuccinimide (sulfoNHS)³ p-Azidobenzoyl hydrazide (ABH) ⁴ Reacts with sugar groups 11.9 A ¹Reactive toward primary amines, sulfhydryls ² Reactive toward primaryamines ³ Reactive toward primary amines, carboxyl groups ⁴ Reactivetoward carbohydrates, nonselective

Hetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond, for example, sulfosuccinimidyl-2-(p-azidosalicylamido)-ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidylgroup reacts with primary amino groups and the phenylazide (uponphotolysis) reacts non-selectively with any amino acid residue.

Other useful cross-linkers, not considered to contain or generate aprotected disulfide, include SATA, SPDP and 2-iminothiolane. The use ofsuch cross-linkers is well known in the art.

Once conjugated, the conjugate is separated from unconjugated SAg andfusion partner polypeptides and from other contaminants. A large anumber of purification techniques are available for use in providingconjugates of a sufficient degree of purity to render them clinicallyuseful. Purification methods based upon size separation, such as gelfiltration, gel permeation or high performance liquid chromatography,will generally be of most use. Other chromatographic techniques, such asBlue-Sepharose separation, may also be used.

Chemotherapeutic and Other Agents

Chemotherapeutic agents can be used together with intrathecal orintratumoral SAg. They can be administered intrathecally, intratumorallyor parenterally by infusion or injection concomitantly with SAg.Preferably they are given together with SAg after 2-7 weeks of treatmentwith the SAg alone. Anti-cancer chemotherapeutic drugs useful in thisinvention include but are not limited to antimetabolites, anthracycline,vinca alkaloid, anti-tubulin drugs, antibiotics and alkylating agents.Representative specific drugs that can be used alone or in combinationinclude cisplatin (CDDP), adriamycin, dactinomycin, mitomycin,caminomycin, daunomycin, doxorubicin, tamoxifen, taxol, taxotere,vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil(5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate,camptothecin, actinomycin-D, mitomycin C, aminopterin, combretastatin(s)and derivatives and prodrugs thereof.

A variety of chemotherapeutic and pharmacological agents may be givenseparately or conjugated to a therapeutic protein of the invention.Exemplary antineoplastic agents that have been conjugated to proteinsinclude doxorubicin, daunomycin, methotrexate and vinblastine. Moreover,the attachment of other agents such as neocarzinostatin, macromycin,trenimon and α-amanitin has been described. See U.S. Pat. Nos.5,660,827; 5,855,866; and 5,965,132; each incorporated by referenceherein. Those of ordinary skill in the art will know how to selectappropriate agents and doses, although, as disclosed, the doses ofchemotherapeutic drugs are preferably reduced when used in combinationwith SAgs according to the present invention.

Another newer class of drugs also termed “chemotherapeutic agents”comprises inducers of apoptosis. Any one or more of such drugs,including genes, vectors, antisense constructs, siRNA constructs, andribozymes, as appropriate, may be used in conjunction with SAgs.

Other agents useful herein are anti-angiogenic agents, such asangiostatin, endostatin, vasculostatin, canstatin and maspin.

Chemotherapeutic agents are administered as single agents or multidrugcombinations, in full or reduced dosage per treatment cycle. They can beadministered with the intrathecal or intratumoral and optionallyparenteral SAg composition although, under a preferred schedule, thechemotherapeutic agent is administered within 36 hours of the last oftwo to four treatments of SAg compositions administered intrathecally orintratumorally.

The combined use of the SAg compositions with low dose, single agentchemotherapeutic drugs is particularly preferred. The choice ofchemotherapeutic drug in such combinations is determined by the natureof the underlying malignancy. For lung tumors, cisplatin is preferred.For breast cancer, a microtubule inhibitor such as taxotere is thepreferred. For malignant ascites due to gastrointestinal tumors, 5-FU ispreferred. “Low dose” as used with a chemotherapeutic drug refers to thedose of single agents that is 10-95% below that of the approved dosagefor that agent (by the U.S. Food and Drug Administration, FDA). If theregimen consists of combination chemotherapy, then each drug dose isreduced by the same percentage. A reduction of >50% of the FDA approveddosage is preferred although therapeutic effects are seen with dosagesabove or below this level, with minimal side effects.

Tumors to treat with SAgs (±chemotherapeutics) using intratumoralinjection are preferably at least 6 cm³ and visible by x-ray, CT,ultrasound, bronchoscopy, laparoscopy, culdoscopy. Intratumorallocalization of the agent being delivered is facilitated withfluoroscopic, CT or ultrasound guidance. Representative tumors that aretreatable with this approach include but are not limited tohepatocellular carcinoma, lung tumors, brain tumors, head and necktumors and unresectable breast tumors. Multiple tumors at differentsites may be treated by intrathecal or intratumoral SAg.

The chemotherapeutic agent(s) selected for therapy of a particular tumorpreferably is one with the highest response rates against that type oftumor. For example, for non-small cell lung cancer (NSCLC),cisplatin-based drugs have been proven effective. Cisplatin may be givenparenterally or intrattunorally. When given intratumorally, Cisplatin ispreferentially in small volume around 1-4 ml although larger volumes canalso work. The smaller volume is designed to increase the viscosity ofthe Cisplatin containing solution in order to minimize or delay theclearance of the drug from the tumor site. Other agents useful in NSCLCinclude the taxanes (paclitaxel and docetaxel), vinca alkaloids(vinorelbine), antimetabolites (gemcitabine), and camptothecin(irinotecan) both as single agents and in combination with a platinumagent.

The optimal chemotherapeutic agents and combined regimens for all themajor human tumors are set forth in Bethesda Handbook of ClinicalOncology, Abraham J et al., Lippincott William & Wilkins, Philadelphia,Pa. (2001); Manual of Clinical Oncology, Fourth Edition, Casciato, D Aet al., Lippincott William & Wilkins, Philadelphia, Pa. (2000) both ofwhich are herein incorporated in entirety by reference.

In one embodiment, these recommended chemotherapeutic agents are usedalone or combined with other chemotherapeutics in full doses.Alternatively they may be administered parenterally by infusion orinjection in doses 10-95% below the FDA recommended therapeutic dose.For intratumoral administration, the dose of a chemotherapeutic drug orbiologic agent is preferably reduced 10- to 50-fold below theFDA-recommended dose for parenteral administration.

Cisplatin has been widely used to treat cancer, with effective doses of20 mg/m² for 5 days every three weeks for a total of three courses.Preferred dose per treatment for intratumoral use of Cisplatin is 5-10mg whereas for intrathecal use 20-80 mg may be administered.Intratumoral cisplatin may be given every 7-14 days for 10-20 treatmentswhereas intrathecal cisplatin may be given every 2-6 weeks for 10-20treatments. Cisplatin delivered in small volumes, e.g., 5-10 mg/1-5 mlsaline, is extremely viscous and may be retained in a tumor for asustained period, thereby acting like a controlled release drug beingreleased from an inert surface. This is indeed the preferred mode ofadministration of Cisplatin when administered intratumorally with orwithout the SAg. Preferably cisplatin is administered together with theSAg in the same syringe.

Other chemotherapeutic compounds include doxorubicin, etoposide,verapamil, podophyllotoxin, and the like which are administered throughintravenous bolus injections at doses ranging from 25-75 mg/m² at 21 dayintervals for adriamycin, to 35-50 mg/m² for etoposide intravenously.

Other agents and therapies that are operable together with or afterintratumoral SAg include, radiotherapeutic agents, antitumor antibodieswith attached anti-tumor drugs such as plant-, fungus-, orbacteria-derived toxin or coagulant, ricin A chain, deglycosylated ricinA chain, ribosome inactivating proteins, sarcins, gelonin, aspergillin,restricticin, a ribonuclease, a epipodophyllotoxin, diphtheria toxin, orPseudomonas exotoxin. Additional cytotoxic, cytostatic or anti-cellularagents capable of killing or suppressing the growth or division of tumorcells include anti-angiogenic agents, apoptosis-inducing agents,coagulants, prodrugs or tumor targeted forms, tyrosine kinase inhibitors(Siemeister et al., 1998), antisense strategies, RNA aptamers, siRNA andribozymes against VEGF or VEGF receptors (Saleh et al., 1996; Cheng etal., 1996; Ke et al., 1998; Parry et al., 1999; each incorporated hereinby reference).

Any of a number of tyrosine kinase inhibitors are useful whenadministered together with, or after, intratumoral SAg. These include,for example, the 4-aminopyrrolo[2,3-d]pyrimidines (U.S. Pat. No.5,639,757). Further examples of small organic molecules capable ofmodulating tyrosine kinase signal transduction via the VEGF-R2 receptorare the quinazoline compounds and compositions (U.S. Pat. No.5,792,771).

Other agents which may be employed in combination with SAgs are steroidssuch as the angiostatic 4,9(11)-steroids and C²¹-oxygenated steroids(U.S. Pat. No. 5,972,922).

Thalidomide and related compounds, precursors, analogs, metabolites andhydrolysis products (U.S. Pat. Nos. 5,712,291 and 5,593,990) may also beused in combination with SAgs and other chemotherapeutic drugs agents toinhibit angiogenesis. These thalidomide and related compounds can beadministered orally.

Certain anti-angiogenic agents that cause tumor regression may beadministered together with, or after, intratumoral SAg. These includethe bacterial polysaccharide CM101 (currently in clinical trials as ananti-cancer drug) and the antibody LM609. CM101 has been wellcharacterized for its ability to induce neovascular inflammation intumors. CM101 binds to and cross-links receptors expressed ondedifferentiated endothelium that stimulate the activation of thecomplement system. It also initiates a cytokine-driven inflammatoryresponse that selectively targets the tumor. CM101 is a uniquelyantiangiogenic agent that downregulates the expression VEGF and itsreceptors. Thrombospondin (TSP-1) and platelet factor 4 (PF4) may alsobe used together with or after intratumoral SAg. These are bothangiogenesis inhibitors that associate with heparin and are found inplatelet α granules.

Interferons and metalloproteinase inhibitors are two other classes ofnaturally occurring angiogenic inhibitors that can be used together withor after intratumoral SAg. Vascular tumors in particular are sensitiveto interferon; for example, proliferating hemangiomas are successfullytreated with IFNα. Tissue inhibitors of metalloproteinases (TIMPs), afamily of naturally occurring inhibitors of matrix metalloproteases(MMPs), can also inhibit angiogenesis and can be used in combinationwith SAgs.

Pharmaceutical Compositions and Administration

One or more of SAg, SAg homologues, fragments, mutants, fusion proteinsand conjugates (SAg agents) are administered by injection, infusion orinstillation or implanted intratumorally or subcutaneously in acontrolled release formulation. SAg agents are most commonlyadministered intrathecally in patients with malignant intrathecal fluidaccumulation due to primary or metastatic tumors. For example, malignantpleural effusions in patients with lung cancer or metastatic breast,gastric or ovarian cancer. SAg agents may also be administeredintrathecally to patients with intrathecal and parenchymal tumor (e.g.,involvement of pleura and lung parenchyma) but little or no fluidaccumulation in the cavitary space. SAg agents may also be administeredintrathecally to patients without malignant involvement or fluidaccumulation in the cavitary space or its membranes but with primary ormetastatic tumor of the organ (e.g., lung, stomach) and/or lymph nodes.For example, SAg may be administered intrapleurally to patients withprimary lung cancer or lung metastases from other primary tumors (e.g.,breast, ovary, gastric) without malignant involvement of the pleura orpleural space. In each of the above examples, intrathecal administrationof the SAg agents may be administered simultaneously or sequentiallywith one or more of the SAg agents administered intratumorally,intralymphatically or intravenously.

SAg agents are administered every 3-10 days for up to three months.Dosages of SAg agents used for intrathecal, intratumoral, intralymphaticand intravenous administration range from 0.1 pg-1 ng/kg.

SAg agents are also administered intratumorally to stimulate a Tcell-based inflammatory response, including release of tumoricidalcytokines and induction of cytotoxic T cells. The amount of SAg agentadministered to a single tumor site ranges from about 0.05-1 ng/kg bodyweight. The intratumoral dose of a cytotoxic drug administered to thetumor site will generally range from about 0.1 to 500, more usuallyabout 0.5 to 300 mg/kg body weight, depending upon the nature of thedrug, size of tumor, and other considerations.

When used to boost the titer of SAg specific antibodies, SAg agents maybe incorporated in an adjuvant vehicle such as alum or Freund'sincomplete adjuvant. These compositions are administered prior to,during or after intrathecal and/or intratumoral administration of theSAg agent.

They are administered subcutaneously, intramuscularly and intradermallyby injection or infusion in doses ranging from 0.1 pg/kg to 1 ng/kg. Toinduce a maximum immune response, boosters with the SAg agent andvehicle at 1-6 month intervals are given.

The pharmaceutical compositions of the present invention will generallycomprise an effective amount of at least a SAg composition dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Combined therapeutics are also contemplated, and the same type ofunderlying pharmaceutical compositions may be employed for both singleand combined medicaments. The intratumoral composition can beadministered to the tumor by needle or catheter via percutaneous entryor via endoscopy, bronchoscopy, culdoscopy or other modes of directvision including directly at the time of surgery. The composition can belocalized into the tumor with CT and/or ultrasound guidance.

With each drug in each tumor, experience will provide an optimum level.One or more administrations may be employed, depending upon the lifetimeof the drug at the tumor site and the response of the tumor to the drug.Administration may be by syringe, catheter or other convenient meansallowing for introduction of a flowable composition into the tumor.Administration may be every three days, weekly, or less frequent, suchas biweekly or at monthly intervals.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. Veterinary uses are equally included within theinvention and “pharmaceutically acceptable” formulations includeformulations for both clinical and/or veterinary use.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. For human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by U.S. Food and Drug Administration. Supplementary activeingredients can also be incorporated into the compositions.

“Unit dosage” formulations are those containing a dose or sub-dose ofthe administered ingredient adapted for a particular timed delivery. Forexample, exemplary “unit dosage” formulations are those containing adaily dose or unit or daily sub-dose or a weekly dose or unit or weeklysub-dose and the like.

Injectable Formulations

The SAg composition of the present invention are preferably formulatedfor parenteral administration, e.g., introduction by injection, infusionor instillation directly into an affected organ cavity (intrathecaladministration) or tumor (intratumorally). Means for preparing aqueouscompositions that contain the SAg compositions are known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and fluid to theextent that syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

The SAg compositions can be formulated into a sterile aqueouscomposition in a neutral or salt form. Solutions as free base orpharmacologically acceptable salts can be prepared in water.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein), and those that areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, trifluoroacetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

Suitable carriers include solvents and dispersion media containing, forexample, water. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the activeagents in the required amount in the appropriate solvent with various ofthe other ingredients enumerated above, as desired, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle thatcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above.

In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques that yield a powder of the active ingredient,plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the SAg composition admixed with anacceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art as exemplified by Remington's Pharmaceutical Sciences, 16thEd. Mack Publishing Company, 1980, or most recent edition, incorporatedherein by reference. Endotoxin contamination should be kept minimally ata safe level, for example, less that 0.5 ng/mg protein. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by the U.S. Food andDrug Administration. Upon formulation, the therapeutic compositions areadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically effective.

Once in an acceptable pharmaceutical form, SAg are administeredintrathecally including but not limited to intrapleurally,intraperitoneally, intra-pericardially, and/or intratumorally andoptionally intra-lymph node and/or parenterally (e.g., intravenously,intramuscularly, subcutaneously) by injection or infusion. SAg are alsodelivered simultaneously or sequentially via one or more routes, e.g.,intrapleurally and intravenously or intrapleurally, intratumorally andintravenously. SAg are also administered simultaneously or sequentiallyin the same or different vehicles, adjuvants and sustained releaseformulations.

Sustained Release Formulations

SAg formulations are easily administered in a variety of dosage forms,including “slow release” capsules or “sustained release” preparations ordevices. Slow release formulations, generally designed to result in aconstant drug level over an extended period, are used to deliver a SAgcomposition as described herein. Such slow release formulations areimplanted intrathecally or intratumorally. Controlled releaseformulations are prepared using polymers to complex or absorb thetherapeutic compositions—SAgs, SAg homologues, chemotherapeutic agentsor combined formulations of a SAg/homologue and a chemotherapeuticagent(s). The rate of release is regulated by (1) selection ofappropriate macromolecules, for example polyesters, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, and protamine sulfate, (2) the concentration ofthe macromolecules and (3) the method of incorporation of the activeagents into the formulation.

Another method to control the duration of action of the presentcontrolled release preparations is to incorporate the SAgs, SAghomologues and/or chemotherapeutic drugs into particles of a polymericmaterial such as polyesters, polyamino acids, hydrogels, for example,poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol); polylactides(e.g., U.S. Pat. No. 3,773,919); copolymers of L-glutamic acid andγ-ethyl-L-glutamate; non-degradable ethylene-vinyl acetate; degradablelactic acid-glycolic acid copolymers, such as the Lupron Depot™(injectable microspheres of lactic acid-glycolic acid copolymer andleuprolide acetate); and poly-D-(−)-3-hydroxybutyric acid.

Alternatively, instead of incorporating the bioactive/pharmaceuticallyactive agents into polymeric particles, the active agents may rather beentrapped in microcapsules prepared by interfacial polymerization.Examples include hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacrylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,macroemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980 or most recent edition). Nanoparticles consisting of SAg, SAghomologue and/or chemotherapeutic agents are delivered intrathecally orintratumorally via insufflation using a gas or air propellant.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. For example, it is known thatwhen encapsulated antibodies remain in the body for a prolonged period,they may denature or aggregate as a result of exposure to moisture at37° C., thus reducing biological activity. Rational strategies areavailable for stabilization, and they depend on the mechanism involved.For example, if the aggregation mechanism involves intermolecular S—Sbond formation through thio-disulfide interchange, stabilization isachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,developing specific polymer matrix compositions, and the like.

A particularly attractive sustained release preparation for use hereincomprises collagen and an effective amount of SAg (or homologue) and acytotoxic drug, as described by Luck et al., RE35,748 and Roskos et al.,U.S. Pat. No. 6,077,545. More detail on preparation is given in Example2.

The collagen composition can be used in the treatment of a wide varietyof tumors including carcinomas, sarcomas and melanomas. Specific typesof tumors include such basal cell carcinoma, squamous cell carcinoma,melanoma, soft tissue sarcoma, solar keratoses, Kaposi's sarcoma,cutaneous malignant lymphoma, Bowen's disease, Wilm's tumor, hepatomas,colorectal cancer, brain tumors; mycosis fungoides, Hodgkin's lymphoma,polycythemia vera, chronic granulocytic leukemia, lymphomas, oat cellsarcoma, etc. The collagen and other composition will be administered toa tumor to provide a cytotoxic amount of drug at the tumor site. Theamount of cytotoxic drug administered to the tumor site will generallyrange from about 0.1 to 500 mg/kg body weight, more usually about 0.5 to300 mg/kg, depending upon the nature of the drug, size of tumor, andother considerations. Vasoconstrictive agents will generally be presentin from 1 to 50% (w/w) of the therapeutic agent. In view of the widediversity of tumors, nature of tumors, effective concentrations of drug,relative mobility and the like, a definitive range cannot be specified.With each drug in each tumor, experience will provide an optimum level.One or more rounds of administration may be employed, depending upon thelifetime of the drug at the tumor site and the response of the tumor tothe drug. Administration may be by syringe, catheter or other convenientmeans allowing for introduction of a flowable composition into thetumor. Administration may be every three days, weekly, or less frequent,such as biweekly or at monthly intervals.

Illustrative of the manner of sustained administration would beadministration of cis-diaminodichloroplatinum (CDDP). Drugconcentrations in the sustained release preparation may vary from 0.01to 50 mg/ml. Injection may be at one or more sites depending on the sizeof the lesion. Needles of about 1-2 mm diameter are convenient. Formultiple injection, templates with predrilled holes may be employed. Thedrug dose will normally be less than 100 mg/m² body surface area.

The present invention is particularly advantageous against those tumorsor lesions that are clinically relevant because of high frequency. Thecompositions provide therapeutic gain with tumors greater than 100 mm³,more particularly, greater than 150 mm³, in volume.

Administration by controlled release of SAg and/or a chemotherapeuticdrug may be used advantageously in conjunction with other forms oftherapy. The tumors or lesions may be irradiated prior and/or subsequentto SAg administration by controlled release. Dose rates may vary fromabout 20 to 250 rad/min, usually 50 to 150 rad/min, depending on thelesion, period of exposure, and the like. Hyperthermia (heat) may beused as an adjunctive treatment. Treatment will usually involve heatingthe tumor and its surrounding tissue to a temperature of about 43° forbetween about 5 and 100 min.

Intratumoral Administration

A SAgs and SETs or a biologically active homologue, fragment or fusionpolypeptide or conjugate as described herein is used for directintratumoral treatment of a tumor mass. SAgs include Staphylococcalenterotoxins A, B, C, D, E, F, 0, H, I, J, K, L, M, YPM, M. arthritides,C. perfringens exotoxin for direct intratumoral treatment of tumormasses. Tumor mass may be those appearing in any organ, palpated orvisualized on x-ray, CT scan, MRI or ultrasound. Intratumoraladministration may be performed with fluoroscopic, CT or ultrasoundguidance.

For intratumoral administration, the dose of a chemotherapeutic drug orbiologic agent is preferably reduced 10- to 50-fold below theFDA-recommended dose for parenteral administration. As noted above, apreferred dose of intratumoral Cisplatin is 5-10 mg in 1-5 ml every 7-14days for 10-40 treatments. This regimen for intratumoral Cisplatin (withor without SAg) is preferred. Preferably Cisplatin is administeredtogether with the SAg in one syringe.

The SAg is dissolved in a conventional vehicle such as saline or it maybe incorporated into a controlled release formulation (mixture orsuspension) preferably biodegradable. All of the biocompatible andbiodegradable and controlled release formulations described herein areuseful. These formulations also include but are not limited to, ethylenevinyl acetate (EVAc: Elvax 40W, Dupont), bioerodible polyanhydrides,polyimino carbonate, sodium alginate microspheres and hydrogels. Dosagesused range from 1 ng to 10 mg. The poly-(D-, L- or DL-lacticacid/polyglycolide) copolymers are preferred.

For intratumoral administration, the SAg composition is preferablyadministered once weekly, and this schedule is continued until the tumorhas shrunk significantly. Generally 3-10 treatments are sufficient. Insome cases the tumor may appear to expand in size during suchintratumoral SAg therapy. This is a result of SAg-stimulatedaccumulation of inflammatory cells and edema. Despite this enlargement,histological examination of such tumors performed during this phaseshows evident tumoricidal effects with inflammatory cell infiltrates.

In the case of an enlarging tumor or a slowly regressing tumor when SAgtherapy is given alone, conventional chemotherapy may be administered topromote tumor killing. A chemotherapeutic agent is preferablyadministered intratumorally alone or together with SAg. Importantly, thechemotherapeutic agent should be given in doses well below thoseprescribed for systemic use of the same agent. Preferably, intratumoralchemotherapy will comprise use of a selected single agent which is knownin the art to be effective against a particular tumor. Moreover,intratumoral combination chemotherapy wherein each agent is given in areduced dose can also be used. Full-dose or reduced-dose systemicchemotherapy can also be used together with, or shortly after,intratumoral SAg therapy. As with intrathecal administration describedherein, intratumoral delivery may be carried out in an outpatientsetting as it requires no hospitalization.

The intratumoral therapy with a SAg and or a SAg homologue can be usedto treat of a wide variety of neoplastic lesions. Indeed, an improvementin 5-year survival from 16% to 26% of small cell lung cancer wasproduced by increase in local control accomplished by altering thefractionation of radiation therapy (Turisi et al., N. Eng. J. Med. 340:265-270 (1999)). Illustrative tumors amenable to intratumoral therapywith SAgs include carcinomas, sarcomas and melanomas, including such asbasal cell carcinoma, squamous cell carcinoma, soft tissue sarcoma,solar keratosis, Kaposi's sarcoma, cutaneous malignant lymphoma, Bowen'sdisease, Wilm's tumor, neuroblastoma, gliomas astrocytomas, hepatoma,colorectal cancer, brain tumors, mycosis fungoides, Hodgkin's lymphoma,polycythemia vera, chronic granulocytic leukemia, lymphomas, oat cellsarcoma, breast carcinoma etc. The intratumoral SAg has particularadvantage for tumors or lesions which are among the most importantclinically because of their frequency. The compositions and methodsdisclosed herein provide therapeutic gain with tumors exceeding 100 mm³in volume, even tumors exceeding 150 mm³.

Superantigens with Radiation Therapy

Local radiation to tumor sites or the mediastinum using the traditionalstandard dose of 60-65 gy may be given concomitant with intrathecal orintratumoral SAg. The radiotherapy may be also be given before or afterthe SAg therapy but in either case there should be a hiatus of no morethan 30 days between the start of SAg therapy and the start orconclusion of radiotherapy. The median survival of patients given thistype of radiotherapy alone is 5% at one year whereas the combinedmodality improves the median survival to more than two years.

Tumor Models and Procedures for Evaluating Anti-Tumor Effects Studies

The various SAg compositions described herein are tested for therapeuticefficacy in several well established rodent models which are consideredto be highly representative of a broad spectrum of human tumors. Theseapproaches are described in detail in Geran, R. I. et al., “Protocolsfor Screening Chemical Agents and Natural Products Against Animal Tumorsand Other Biological Systems (Third Edition)”, Canc. Chemother. Reports,Pt 3, 3:1-112, which is hereby incorporated by reference in itsentirety.

A. Calculation of Mean Survival Time (MST)

MST (days) is calculated according to the formula:

$\frac{S + {AS}_{({A - 1})} - {\left( {B + 1} \right){NT}}}{S_{({A - 1})} - {NT}}$

-   Day: Day on which deaths are no longer considered due to drug    toxicity. For example, with treatment starting on Day 1 for survival    systems (such as L1210, P388, B16, 3LL, and W256): Day A=Day 6; Day    B=Day beyond which control group survivors are considered    “no-takes.”-   S: If there are “no-takes” in the treated group, S is the sum from    Day A through Day B. If there are no “no-takes” in the treated    group, S is the sum of daily survivors from Day A onward.-   S(A−1): Number of survivors at the end of Day (A−1).-   Example: for 3LE21, S(A−1)=number of survivors on Day 5.-   NT: Number of “no-takes” according to the criteria given in    Protocols 7.300 and 11.103.

B. TIC Computed for all Treated Groups

$\text{T/C} = {\frac{{MST}\mspace{14mu} {of}\mspace{14mu} {treated}\mspace{14mu} {group}}{{MST}\mspace{14mu} {of}\mspace{14mu} {control}\mspace{14mu} {group}} \times 100}$

Treated group animals surviving beyond Day Bare eliminated fromcalculations (as follows):

No. of survivors in treated Percent of “no-takes” group beyond Day B incontrol group Conclusion 1 Any percent “no-take” 2 <10 drug inhibition³10 “no-takes” ³3 <15 drug inhibitions ³15 “no-takes”

Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, allsurvivors on Day B are used in the calculation of TIC for the positivecontrol. Surviving animals are evaluated and recorded on the day ofevaluation as “cures” or “no-takes.”

Calculation of Median Survival Time (MedST)

MedST is the median day of death for a test or control group. If deathsare arranged in chronological order of occurrence (assigning tosurvivors, on the final day of observation, a “day of death” equal tothat day), the median day of death is a day selected so that one half ofthe animals died earlier and the other half died later or survived. Ifthe total number of animals is odd, the median day of death is the daythat the middle animal in the chronological arrangement died. If thetotal number of animals is even, the median is the arithmetical mean ofthe two middle values. Median survival time is computed on the basis ofthe entire population and there are no deletion of early deaths orsurvivors, with the following exception:

C. Computation of MedST From Survivors

If the total number of animals including survivors (N) is even, theMedST in days is (X+Y)/2, where X is the earliest day when the number ofsurvivors is N/2, and Y is the earliest day when the number of survivors(N/2)−1. If N is odd, the MedST is X.

D. Computation of MedST From Mortality Distribution

If the total number of animals including survivors (N) is even, theMedST in days is (X+Y)/2, where X is the earliest day when thecumulative number of deaths is N12, and Y is the earliest day when thecumulative number of deaths is (N/2)+1. If N is odd, the MedST is X.“Cures” and “no-takes” in systems evaluated by MedST are based upon theday of evaluation. On the day of evaluation any survivor not considereda “no-take” is recorded as a “cure.” Survivors on day of evaluation arerecorded as “cures” or “no-takes,” but not eliminated from thecalculation.

E. Calculation of Approximate Tumor Weight from Measurement of TumorDiameters with Vernier Calipers

The use of diameter measurements (with Vernier calipers) for estimatingtreatment effectiveness on local tumor size permits retention of theanimals for lifespan observations. When the tumor is implanted sc, tumorweight is estimated from tumor diameter measurements as follows. Theresultant local tumor is considered a prolate ellipsoid with one longaxis and two short axes. The two short axes are assumed to be equal. Thelongest diameter (length) and the shortest diameter (width) are measuredwith Vernier calipers. Assuming specific gravity is approximately 1.0,and rounding π to 3, the tumor mass (in mg) is calculated by multiplyingthe length of the tumor (in mm) by the width squared and dividing theproduct by two:

${{Tumor}\mspace{20mu} {{weight}({mg})}} = {\frac{({length}) \times ({width})^{2}}{2}\mspace{14mu} {or}\mspace{14mu} \frac{L \times (W)^{2}}{2}}$

The reporting of tumor weights calculated in this way is acceptableinasmuch as the assumptions result in as much accuracy as theexperimental method warrants.

F. Calculation of Tumor Diameters

The effects of a drug on the local tumor diameter may be reporteddirectly as tumor diameters without conversion to tumor weight. Toassess tumor inhibition by comparing the tumor diameters of treatedanimals with the tumor diameters of control animals, the three diametersof a tumor are averaged (the long axis and the two short axes). A tumordiameter T/C of 75% or less indicates activity and a TIC of 75% isapproximately equivalent to a tumor weight T/C of 42%.

G. Calculation of Mean Tumor Weight From Individual Excised Tumors

The mean tumor weight is defined as the sum of the weights of individualexcised tumors divided by the number of tumors. This calculation ismodified according to the rules listed below regarding “no-takes.” Smalltumors weighing 39 mg or less in control mice or 99 mg or less incontrol rats, are regarded as “no-takes” and eliminated from thecomputations. In treated groups, such tumors are defined as “no-takes”or as true drug inhibitions according to the following rules:

Percent of Percent of small tumors in “no-takes” in treated groupcontrol group Action ≦17 Any percent no-take; not used in calculations18-39 <10 drug inhibition; use in calculations ≧10 no-takes; not used incalculations ≧40 <15 drug inhibition; use in calculations ≧15 Code allnontoxic tests “33”

Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, thetumor weights of all surviving animals are used in the calculation ofT/C for the positive control (TIC defined above) SDs of the mean controltumor weight are computed the factors in a table designed to estimate SDusing the estimating factor for SD given the range (difference betweenhighest and lowest observation). Biometrik Tables for Statisticians(Pearson E S, and Hartley H G, eds.) Cambridge Press, Vol. 1, Table 22,p. 165.

II. Specific Tumor Models A. Lymphoid Leukemia L1210

Summary: Ascitic fluid from donor mouse is transferred into recipientBDF1 or CDF1 mice. Treatment begins 24 hours after implant. Results areexpressed as a percentage of control survival time. Under normalconditions, the inoculum site for primary screening is i.p., thecomposition being tested is administered i.p., and the parameter is meansurvival time. Origin of tumor line: induced in 1948 in spleen and lymphnodes of mice by painting skin with MCA. J Natl Cancer Inst. 13:1328,1953.

Animals One sex used for all test and control animals in one experiment.Tumor Transfer Inject ip, 0.1 ml of diluted ascitic fluid containing 10⁵cells Propagation DBA/2 mice (or BDF1 or CDF1 for one generation). Timeof Transfer Day 6 or 7 Testing BDF₁ (C57BL/6 × DBA/2) or CDF₁ (BALB/c ×DBA/2) Time of Transfer Day 6 or 7 Weight Within a 3-g range, minimumweight of 18 g for males and 17 g for females. Exp Size (n) 6/group; No.of control groups varies according to number of test groups.

Testing Schedule

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive 1 μg of the test composition in 0.5 ml saline.Controls receive saline alone. Treatment is one dose/week. Any survivingmice are sacrificed after 4 wks of therapy. 5 Weigh animals and record.20 If there are no survivors except those treated with positive controlcompound, evaluate 30 Kill all survivors and evaluate experiment.Quality Control (“QC”): Acceptable control survival time is 8-10 days.Positive control compound is 5-fluorouracil; single dose is 200mg/kg/injection, intermittent dose is 60 mg/kg/injection, and chronicdose is 20 mg/kg/injection. Ratio of tumor to control (TIC) lower limitfor positive control compound is 135%.Evaluation: Compute mean animal weight on Days 1 and 5, and at thecompletion of testing compute T/C for all test groups with >65%survivors on Day 5. A TIC value 85% indicates a toxic test. An initialT/C 125% is considered necessary to demonstrate activity. A reproducedT/C 125% is considered worthy of further study. For confirmed activity acomposition should have two multi-dose assays that produce a T/C 125%.

B. Lymphocytic Leukemia P388

Summary: Ascitic fluid from donor mouse is implanted in recipient BDF1or CDF1 mice. Treatment begins 24 hours after implant. Results areexpressed as a percentage of control survival time. Under normalconditions, the inoculum site for primary screening is ip, thecomposition being tested is administered ip daily for 9 days, and theparameter is MedST. Origin of tumor line: induced in 1955 in a DBA/2mouse by painting with MCA. Scientific Proceedings, Pathologists andBacteriologists 33:603, 1957.

Animals One sex used for all test and control animals in one experiment.Tumor Transfer Inject ip, 0.1 ml of diluted ascitic fluid containing 10⁶cells Propagation DBA/2 mice (or BDF1 or CDF1 for one generation). Timeof Transfer Day 7 Testing BDF₁ (C57BL/6 × DBA/2) or CDF₁ (BALB/c ×DBA/2) Time of Transfer Day 6 or 7 Weight Within a 3-g range, minimumweight of 18 g for males and 17 g for females. Exp Size (n) 6/group; No.of control groups varies according to number of test groups.

Testing Schedule

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive 1 μg of the test composition in 0.5 ml saline.Controls receive saline alone. Treatment is one dose/week. Any survivingmice are sacrificed after 4 wks of therapy. 5 Weigh animals and record.20 If there are no survivors except those treated with positive controlcompound, evaluate 30 Kill all survivors and evaluate experiment.Acceptable MedST is 9-14 days. Positive control compound is5-fluorouracil: single dose is 200 mg/kg/injection, intermittent dose is60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. TIC lowerlimit for positive control compound is 135% Check control deaths, notakes, etc.QCI: Acceptable MedST is 9-14 days. Positive control compound is5-fluorouracil: single dose is 200 ma/kg/injection, intermittent dose is60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. T/C lowerlimit for positive control compound is 135%. Check control deaths, notakes, etc.Evaluation: Compute mean animal weight on Days 1 and 5, and at thecompletion of testing compute T/C for all test groups with >65%survivors on Day 5. A T/C value of 85% indicates a toxic test. Aninitial T/C of 125% is considered necessary to demonstrate activity. Areproduced T/C 125% is considered worthy of further study. For confirmedactivity a composition should have two multi-dose assays that produce aT/C 125%.

C. Melanotic Melanoma B16

Summary: Tumor homogenate is implanted ip or sc in BDF1 mice. Treatmentbegins 24 hours after either ip or sc implant or is delayed until an sctumor of specified size (usually approximately 400 mg) can be palpated.Results expressed as a percentage of control survival time. Thecomposition being tested is administered ip, and the parameter is meansurvival time. Origin of tumor line: arose spontaneously in 1954 on theskin at the base of the ear in a C57BL/6 mouse. Handbook on GeneticallyStandardized fax Mice. Jackson Memorial Laboratory, Bar Harbor, Me.,1962. See also Ann NY Acad Sci 100, Parts 1 and 2, 1963.

Animals One sex used for all test and control animals in one experiment.Propagation Strain C57BL/6 mice Tumor Transfer Implant fragment sc bytrochar or 12-g needle or tumor homogenate* every 10-14 days intoaxillary region with puncture in inguinal region. Testing Strain BDF₁(C57BL/6 × DBA/2) Time of Transfer Excise sc tumor on Day 10-14 fromdonor mice and implant as above Weight Within a 3-g range, minimumweight of 18 g for males and 17 g for females. Exp Size (n) 10/group;No. of control groups varies according to number of test groups. *Tumorhomogenate: Mix 1 g or tumor with 10 ml of cold balanced salt solution,homogenize, and implant 0.5 ml of tumor homogenate ip or sc. Fragment: A25-mg fragment may be implanted sc.

Testing Schedule

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive 1 μg of the test composition in 0.5 ml saline.Controls receive saline alone. Treatment is one dose/week. Any survivingmice are sacrificed after 8 wks of therapy. 5 Weigh animals and record.60 Kill all survivors and evaluate experiment.QC: Acceptable control survival time is 14-22 days. Positive controlcompound is 5-fluorouracil: single dose is 200 mg/kg/injection,intermittent dose is 60 mg/kg/injection, and chronic dose is 20mg/kg/injection. TIC lower limit for positive control compound is 135%Check control deaths, no takes, etc.Evaluation: Compute mean animal weight on Days 1 and 5, and at thecompletion of testing compute T/C for all test groups with >65%survivors on Day 5. A T/C value of 85% indicates a toxic test. Aninitial T/C of 125% is considered necessary to demonstrate activity. Areproduced T/C 125% is considered worthy of further study. For confirmedactivity a composition should have two multi-dose assays that produce aT/C 125%.

Metastasis after IV Injection of Tumor Cells

10⁵ B16 melanoma cells in 0.3 ml saline are injected intravenously inC57BL/6 mice. The mice are treated intravenously with 1 g of thecomposition being tested in 0.5 ml saline. Controls receive salinealone. The treatment is given as one dose per week. Mice sacrificedafter 4 weeks of therapy, the lungs are removed and metastases areenumerated.

C. 3LL Lewis Lung Carcinoma

Summary: Tumor may be implanted sc as a 2-4 mm fragment, or im as a2×10⁶-cell inoculum. Treatment begins 24 hours after implant or isdelayed until a tumor of specified size (usually approximately 400 mg)can be palpated. The composition being tested is administered ip dailyfor 11 days and the results are expressed as a percentage of thecontrol. Origin of tumor line: arose spontaneously in 1951 as carcinomaof the lung in a C57BL/6 mouse. Cancer Res 15:39, 1955. See, alsoMalave, I. et al., J. Nat'l. Canc. Inst. 62:83-88 (1979).

Animals One sex used for all test and control animals in one experiment.Propagation Strain C57BL/6 mice Tumor Transfer Inject cells im in hindleg or implant fragment sc in axillary region with puncture in inguinalregion. Transfer on day 12-14 Testing Strain BDF₁ (C57BL/6 × DBA/2) orC3H mice Time of Transfer Same as above Weight Within a 3-g range,minimum weight of 18 g for males and 17 g for females. Exp Size (n)6/group for sc implant, or 10/group for im implant.; No. of controlgroups varies according to number of test groups.

Testing Schedule

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive 1 μg of the test composition in 0.5 ml saline.Controls receive saline alone. Treatment is one dose/week. Any survivingmice are sacrificed after 4 wks of therapy. 5 Weigh animals and record.Final day Kill all survivors and evaluate experiment.QC: Acceptable im tumor weight on Day 12 is 500-2500 mg. Acceptable imtumor MedST is 18-28 days. Positive control compound iscyclophosphamide: 20 mg/kg/injection, qd., Days 1-11. Check controldeaths, no takes, etc.Evaluation: Compute mean animal weight when appropriate, and at thecompletion of testing compute T/C for all test groups. When theparameter is tumor weight, a reproducible T/C of 42% is considerednecessary to demonstrate activity. When the parameter is survival time,a reproducible T/C of 125% is considered necessary to demonstrateactivity. For confirmed activity a composition must have two multi-doseassays

D. 3LL Lewis Lung Carcinoma Metastasis Model

This model has been utilized by a number of investigators. See, forexample, Gorelik, E. et al., J. Nat'l. Canc. Inst. 65:1257-1264 (1980);Gorelik, E. et al., Rec. Results Canc. Res. 75:20-28 (1980); Isakov, N.et al., Invasion Metas. 2:12-32 (1982) Talmadge I. E. et al., J. Nat'l.Canc. Inst. 69:975-980 (1982); Hilgard, P. et al., Br. J. Cancer35:78-86 (1977)).

Mice: male C57BL/6 mice, 2-3 months old. Tumor: The 3LL Lewis LungCarcinoma was maintained by se transfers in C57BL/6 mice. Following se,im or intra-footpad transplantation, this tumor produces metastases,preferentially in the lungs. Single-cell suspensions are prepared fromsolid tumors by treating minced tumor tissue with a solution of 0.3%trypsin. Cells are washed 3 times with PBS (pH 7.4) and suspended inPBS. Viability of the 3LL cells prepared in this way is generally about95-99% (by trypan blue dye exclusion). Viable tumor cells (3×10⁴-5×10⁶)suspended in 0.05 ml PBS are injected into the right hind foot pads ofC57BL/6 mice. The day of tumor appearance and the diameters ofestablished tumors are measured by caliper every two days. Typically,mice receive 1 μg of the composition being tested in 0.5 ml saline.Controls receive saline alone. The treatment is given as one or twodoses per week.

In experiments involving tumor excision, mice with tumors 8-10 mm indiameter are divided into two groups. In one group, legs with tumors areamputated after ligation above the knee joints. Mice in the second groupare left intact as nonamputated tumor-bearing controls. Amputation of atumor-free leg in a tumor-bearing mouse has no known effect onsubsequent metastasis, ruling out possible effects of anesthesia, stressor surgery. Surgery is performed under Nembutal anesthesia (60 mgveterinary Nembutal per kg body weight).

Determination of Metastasis Spread and Growth

Mice are killed 10-14 days after amputation. Lungs are removed andweighed. Lungs are fixed in Bouin's solution and the number of visiblemetastases is recorded. The diameters of the metastases are alsomeasured using a binocular stereoscope equipped with amicrometer-containing ocular under 8× magnification. On the basis of therecorded diameters, it is possible to calculate the volume of eachmetastasis. To determine the total volume of metastases per lung, themean number of visible metastases is multiplied by the mean volume ofmetastases. To further determine metastatic growth, it is possible tomeasure incorporation of ¹²⁵IdUrd into lung cells (Thakur, M. L. et al,J. Lab. Clin. Med. 89:217-228 (1977). Ten days following tumoramputation, 25 mg of FdUrd is inoculated into the peritoneums oftumor-bearing (and, if used, tumor-resected mice. After 30 min, mice aregiven 1 mCi of ¹²⁵IdUrd. One day later, lungs and spleens are removedand weighed, and a degree of ¹²⁵IdUrd incorporation is measured using agamma counter.

Statistics: Values representing the incidence of metastases and theirgrowth in the lungs of tumor-bearing mice are not normally distributed.Therefore, non-parametric statistics such as the Mann-Whitney U-Test maybe used for analysis.

Study of this model by Gorelik et al. (1980, supra) showed that the sizeof the tumor cell inoculum determined the extent of metastatic growth.The rate of metastasis in the lungs of operated mice was different fromprimary tumor-bearing mice. Thus in the lungs of mice in which theprimary tumor had been induced by inoculation of large doses of 3LLcells (1-5×10⁶) followed by surgical removal, the number of metastaseswas lower than that in nonoperated tumor-bearing mice, though the volumeof metastases was higher than in the nonoperated controls. Using¹²⁵IdUrd incorporation as a measure of lung metastasis, no significantdifferences were found between the lungs of tumor-excised mice andtumor-bearing mice originally inoculated with 10⁶ 3LL cells. Amputationof tumors produced following inoculation of 10⁵ tumor cells dramaticallyaccelerated metastatic growth. These results were in accord with thesurvival of mice after excision of local tumors. The phenomenon ofacceleration of metastatic growth following excision of local tumors hadbeen observed by other investigators. The growth rate and incidence ofpulmonary metastasis were highest in mice inoculated with the lowestdoses (3×10⁴-10⁵ of tumor cells) and characterized also by the longestlatency periods before local tumor appearance. Immunosuppressionaccelerated metastatic growth, though nonimmunologic mechanismsparticipate in the control exerted by the local tumor on lung metastasisdevelopment. These observations have implications for the prognosis ofpatients who undergo cancer surgery.

E. Walker Carcinosarcoma 256

Summary: Tumor may be implanted sc in the axillary region as a 2-6 mmfragment, im in the thigh as a 0.2-nil inoculum of tumor homogenatecontaining 10⁶ viable cells, or ip as a 0.1-ml suspension containing 10⁶viable cells. Treatment of the composition being tested is usually ip.Origin of tumor line: arose spontaneously in 1928 in the region of themammary gland of a pregnant albino rat. J Natl Cancer Inst 13:1356,1953.

Animals One sex used for all test and control animals in one experiment.Propagation Strain Random-bred albino Sprague-Dawley rats Tumor TransferS.C. fragment implant is by trochar or 12-g needle into axillary regionwith puncture in inguinal area. I.m. implant is with 0.2 ml of tumorhomogenate (containing 10⁶ viable cells) into the thigh. I.p. implant iswith 0.1 ml suspension (containing 10⁶ viable cells) Day 7 for im or ipimplant; Days 11-13 for sc implant Testing Strain Fischer 344 rats orrandom-bred albino rats Time of Transfer Same as above Weight 50-70 g(maximum of 10-g weight range within each experiment) Exp Size (n)6/roup; No. of control groups varies according to number of test groups.

Test Prepare drug Administer drug Weigh animals Evaluate system on day:on days: on days on days 5WA16 2 3-6 3 and 7 7 5WA12 0 1-5 1 and 5 10-145WA31 0 1-9 1 and 5 30In addition the following general schedule is followed

DAY PROCEDURE 0 Implant tumor. Prepare materials. Run positive controlin every odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive 1 μg of the test composition in 0.5 ml saline.Controls receive saline alone. Treatment is one dose/week. Any survivingmice are sacrificed after 4 wks of therapy. Final day Kill all survivorsand evaluate experiment.QC: Acceptable i.m. tumor weight or survival time for the above threetest systems are: 5WA16: 3-12 g.; 5WA12: 3-12 g.; 5WA31 or 5WA21: 5-9days.Evaluation: Compute mean animal weight when appropriate, and at thecompletion of testing compute T/C for all test groups. When theparameter is tumor weight, a reproducible T/C 42% is considerednecessary to demonstrate activity. When the parameter is survival time,a reproducible T/C 125% is considered necessary to demonstrate activity.For confirmed activity

F. A20 Lymphoma

10⁶ murine A20 lymphoma cells in 0.3 ml saline are injectedsubcutaneously in Balb/c mice. The mice are treated intravenously with 1g of the composition being tested in 0.5 ml saline. Controls receivesaline alone. The treatment is given as one dose per week. Tumor growthis monitored daily by physical measurement of tumor size and calculationof total tumor volume. After 4 weeks of therapy the mice are sacrificed.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

INTRODUCTION TO EXAMPLES

Prior to the present invention, those skilled in the art were notinclined to consider administering SAgs intrathecally into the pleuralspace in patients with MPE (or intratumorally into patients withmalignant lung or brain nodules.) This is because published studies(Terman et cal., et al., N. Engl. J. Med. 305:1195-2000 (1981); Young etal., Am. J. Med. 75:278-88 (1983)) had shown that SEB (together withprotein A) administration to patients with metastatic breast cancerresulted in severe pulmonary toxicity which manifested as objectivelyconfirmed acute respiratory distress syndrome (ARDS) with hypoxemia (dueto non-cardiogenic pulmonary edema). The hypoxemia was worse in apatient with preexisting metastatic lung tumor who also developed severebronchospasm and a large pleural effusion requiring repeatedthoracenteses. This strong reaction prompted the above authors to warnthat SEB treatment should not be carried out in patients with pulmonarymetastases (Terman, D S CRC Crit. Rev. Oncol. Hematol. 4:103-24 (1985)).Moreover, pathology studies of primates infused with SEB showed atendency for the protein to localize in the pulmonary vasculatureinjuring endothelial cells and causing pulmonary edema (Finegold M J,Lab. Invest. 16:912-924 (1967)). Based on the foregoing, a person ofordinary skill in the art would have concluded that administration of aSAg directly into the pleural space to treat an MPE was contraindicatedbecause it was liable to exacerbate the effusion and induce lifethreatening hypoxemia and bronchospasm.

Surprisingly, as presented below, the present inventor discovered that,notwithstanding the earlier results cited above that taught away fromintrapleural and intratumoral administration of SAgs, intrathecaladministration and intratumoral administration of SEs directly into thepleural space resulted in successful treatment of patients with MPE(intrathecal=intrapleural) and disappearance of a large lung carcinoma(intratumoral). Indeed, all twelve of the first patients with MPEtreated in this manner showed complete regressions of their pleuraleffusions with minimal toxicity. A patient treated with a large lungadenocarcinoma treated with intratumoral SAg and low dose cisplatinshowed a complete regression of his tumor. Importantly, Examples 1 and 2prove that a native SAg, not combined with a tumor specific antibody asis taught in the prior art, is an effective antitumor therapeutic agent.

Example 1

Intrathecal (Intrapleural) Injection of SEC in Patients with MalignantPleural Effusions

Methods

Patients: From February 1999 to October 2002, 14 consecutive patientswith NSCLC and MPE were evaluated. Twelve were considered to beevaluable for response and ten for survival. Patins were required tohave NSCLC with at least a 350 ml effusion. Chemotherapy, radiation andall other biological response modifying agents with antitumor activitieswere discontinued at least a month before starting treatment. Patientsthat had been treated with prior intrapleural chemotherapy, pleurodesis,lobectomy or pneumonectomy on the affected side were excluded. Pleuraleffusions were diagnosed by chest radiographs, chest CT andultrasonography. The diagnosis of MPE was established by positivepleural fluid cytology. In two patients, cytology of pleural fluidexamined 24 hours after treatment showed evidence of tumor killing.

Before each course of treatment, the patients received a completephysical examination, ECG, respiratory function tests, CBC, serumbiochemistry tests, and urinalysis. Each patient had a chest radiogramand sonogram of the pleural fluid before starting treatment to documentthe presence of pleural fluid. Blood and pleural effusion samples wereobtained by venipuncture and thoracentesis, respectively, before and 6hours after selected procedures. Chest radiograms and sonograms wereobtained for every case monthly until the completion of the study.Patients were also screened continuously for side effects which weregraded according to the World Health Organization toxicity scale.Palliative radiotherapy was permitted as long as it was not directed atthe involved pleura. Karnofsky performance status (KPS) was recorded forall patients but was not a criterion for eligibility in the trial.

Treatment: Staphylococcal enterotoxin C (SEC) 2 ng/ml was used. SECinduced mitogenesis in peripheral blood mononuclear cell in doses below10 pg/ml. Thoracentesis was performed after sonographic localization ofthe pleural effusion. Once MPE was demonstrated, thoracentesis wascarried out with complete removal of the pleural effusion. With thepatient in the sitting position, the entry site was cleaned with asepticsolution and the skin infiltrated with 2% lidocaine. The needle wasdirected into the intercostal space just above the rib. A small skinincision was made, and an 18-gauge needle was introduced into thepleural space. Fluid was withdrawn through a three-way stopcock.Following thoracentesis and complete evacuation of the pleural effusion,SEC at 20 to 40 nanograms in 10-20 ml of diluent were gently injectedinto the pleural cavity through the same thoracentesis needle. Patientswere instructed to rest for 24 hours and were monitored for theappearance of new symptoms and changes in vital signs.

Patients were followed daily for recurrence of pleural fluid by physicalexamination and ultrasound and chest radiography. Thoracentesis and SECinjection were repeated every 3-7 days until there was no furtherre-accumulation of pleural fluid. In general, patients required anaverage of 3-4 treatments to achieve remission,

Evaluation of Responses: Complete response (CR) was defined as theabsence of pleural fluid re-accumulation confirmed by chest radiographsand sonography without the need for thoracentesis for >4 weeks. Apartial response (PR) was defined as a decrease of ˜50% of pretreatmentpleural effusion volume and no appearance of new effusions over a periodof >4 weeks. Short term responses were recorded at 30 days and long termresponses at 90 days after completing SEC treatment. No response (NR)was defined as less than a 50% decrease in pleural effusion volume.Progressive disease was defined as was at least a 25% increase in theeffusion volume above pretreatment levels.

SEC treatments were continued every 3-7 days until there was nosignificant pleural fluid reaccumulation. The patients were thenobserved monthly for pleural fluid reaccumulation using ultrasoundand/or chest radiography. The following were not considered treatmentfailures: blunting of the costo-diaphragmatic angles, pleural reactionand small amounts of loculated pleural fluid detected on ultrasound thatwas inaccessible to thoracentesis.

Statistical Evaluation of Survival: The S-Plus statistical softwarepackage (Professional Edition 6 for Windows, Insightful Corporation,Seattle, Wash.) was used to evaluate survival based on measurements fromfirst day of enterotoxin treatment. Kaplan Meir survival curves werederived with the survival analysis program developed at the Mayo Clinicand incorporated in the S-Plus package. Estimates of survivalprobabilities, MedST and the variance of MedST were obtained from theSEC treated group and from comparative groups. A log rank test wasperformed comparing the SEC treatment group with a control group and tocompare two different drug administration protocols. The Wilcoxon ranksum statistic was used to test the comparison of Karnofsky scoresbetween two groups. The relation of the level of pleural effusion tosurvival was analyzed using the Cox proportional hazards model.Hematologic Studies Complete blood counts in the peripheral blood andpleural fluid were obtained before and 6-24 hours after SEC treatment inall patients.

Results

Patient Characteristics: Fourteen consecutive patients with NSCLC wereconsidered for the study, 12 of which were evaluable for response and 10for survival. Two patients were excluded from data for response andsurvival. The first had received intrapleural chemotherapy before thistreatment and the second had a prior pneumonectomy on the affected side.Two additional patients were evaluable for response but were lost tolong-term followup (therefore unevaluable for survival). Patientdemographics are shown in Table 4. Of the 12 evaluable patients all weremales, median age was 68 years (46-82). The NSCLC consisted of 9adenocarcinomas and 3 squamous cell carcinomas. Pleural effusions wereunilateral in 11 patients, bilateral in one and associated with ascitesand pericardial effusion in two and one patient respectively. The medianinitial volume of the pleural effusions was 575 ml (350-1100 ml). Themedian pretreatment Karnofsky performance status (KPS) was 45 (30-60)(Table 4).

Dose, Schedule and Route of Administration

In all patients, SEC was delivered intrapleurally immediately afterthoracentesis in doses ranging from 20-40 ng. In general, the procedurewas repeated every 3-7 days. A total of 45 intrapleural treatments withSSEC were administered. The mean number of IP treatments required beforesignificant fluid reaccumulation ceased was 3.8±1.3. Along withintrapleural SEC, five patients also received SEC IV daily for 30, 2114, 6 and 3 days respectively commencing at the same time as the firstintrapleural administration of SEC. Tables 5 shows the SEC dosage andduration of remission in each of the 12 evaluable patients.

TABLE 4 Patient Demographics Characteristics Numbers/values No. ofevaluable patients 12 Response 12 Survival 10 Gender all 12 male MeanAge in years (range) 64.8 (46-82)    KPS - mean (range) 45 (20-60) Primary Lung Tumors 12 Non Small Cell Lung Carcinomas (NSCLC) 11Adenocarcinoma  9 Squam. cell carcinoma  3 Mean Initial Size of Effusionin ml (range) 595 (350-1100)

TABLE 5 Recurrence of Malignant Pleural Effusions in Patients AfterIntrapleural SSEC NSCLC Pt # SEC Regimen age/gender Prior Therapy dose,freq., duration Response & Duration After Therapy 1. 82/M Radiotherapy25 ng IP Q1 wk × 3 wk. No recurrent effusion 26 mo. 50 ng IP Q1 wk × 3wk. 10 ng IV QED × 30 days 2. 67/M Radiotherapy 25 ng IP Q1 wk × 4 wk.No recurrent effusion 11 mo. 5 ng IV QED × 21 days 3. 66/M 25 ng IP Q1wk × 4 wk. Minimal effusion 15 Mo 4. 61/M Intrapleural 25 ng IP Q1 wk ×4 wks No recurrent effusion 7 mo. cytoxan 5 ng IV QD × 14 days 5. 47/M20 ng IP I wk × 3 No recurrent effusion 5 mo (suicide) 6. 73/MRadiotherapy 25 ng IP Q 3-4 days × 5 No recurrent effusion 8 mo. 7. 68/M25 ng IP Q 3-4 days × 4 No recurrent effusion 6 mo 8. 69/M 25 ng IP Q 1wk × 3 No recurrent effusion 7 mo. 9. 56/M 25 ng IP Q 3-4 days × 5 Norecurrent effusion 7 mo 10 ng IV QD × 21 days 10. 65/M 25 ng IP Q 3-4days × 3 No recurrent effusion 5 mos 10 ng IV QD × 14 days 11. 46/M 25ng IP × 1 Minimal pleural fluid 3 mo. Lost to 10 ng IV QD × 3 daysfollowup 12. 78 M 25 ng IP Q3-4 days × 3 No recurrent effusion 5 mo.Lost to followup

Responses: Twelve patients were evaluable for response, i.e., recurrenceof pleural effusion following the last SEC treatment. Eleven had acomplete response and 1 had a partial response. All 12 patients, showedno recurrent effusion more than 90 days after their last SEC treatment.One month after treatment, the median pretreatment KPS of 45 (30-60)improved to a median KPS of 70 (60-90) (p<0.05) in association withresolution of the effusions. In patient #1, a left pleural effusionrecurred 6 month after his first SEC treatment. He was retreated withSEC IP Q 3-4 days×4 after which the effusion resolved and has notrecurred. He has been in a disease free status for 20 months after hislast treatment and is alive 25 months from the first SEC treatment(Table 5). Sixteen months after starting therapy, patient #4 had arecurrent symptomatic pleural and pericardial effusion of moderate sizeon ultrasound. Within two weeks of treatment with two SEC instillationssymptoms remitted as did the pleural and pericardial effusions. However,the patient refused additional treatment and hence the effect of thislimited retreatment could not be evaluated. Effusions did not reappearin patients #2 and #5 until death 11.5 and 8 months respectively afterstarting SEC treatment.

Survival: Survival data is displayed in Table 6. Ten patients wereevaluable for survival. Median pretreatment KPS was 45 (30-60). Themedian survival for the 10 evaluable patients in the SSEC-treated groupwas 8.25 months. This was compared to the median survival of 2.4 monthsfor control group comprising 21 consecutive current control patientswith MPE from NSCC treated with talc pleurodesis at University ofCalifornia San Diego from 1993-1998 (p=0.0096) (FIG. 1). Eleven patientsfrom the latter group with pretreatment KPS and distribution comparableto the SSEC-treated group (p=0.8) had a median survival of 2.4 months(p=0.0014) (FIG. 2). Patients in the SEC-treated group survived on theorder of 3.6 fold longer than talc pleurodesis-treated controls (FIGS. 1& 2). Three patients in the SEC-treated group survived for 350 days ormore with one patient still alive 26 months after starting therapy whileno patients survived longer than 342 days among the 11 patients thetalc-treated control group (FIG. 2). Survival in the SEC group could notbe predicted from pretreatment pleural volume (p=0.15).Application to Other Tumors: SEC was given to intrapleurally to threepatients with MPEs from small cell carcinoma of the lung, uterinesarcoma and melanoma respectively according to the same protocol forNSCLC patients. Two patients (small cell carcinoma and uterine sarcoma)had prior chemotherapy. All three showed complete resolution of theirmalignant effusions lasting 3, 1.5 and 4 months respectively. Toxicitywas minimal.

Hence, in view of the broad range of tumors shown to be responsive tointrathecal SEC, it would be fully expected that the SEC therapy wouldproduce objective anti-tumor effects against substantially allmalignancies irrespective of origin exhibiting a malignant pleuraleffusion.

TABLE 6 Survival of Patients Treated with Intrapleural SuperantigenStaphylococcal Enterotosin C (SEC) NSCLC Pt. # Initial PleuralAge/Gender Fluid Volume KPS * Survival (unique Pt ID) (ml) Pre/post(months) ** 1. 82/M (#175414) 1100 30/90 26 (Alive) 2. 67/M (#167251)350 40/70 11.5 (Dead) 3. 66/M (#181383) 550 50/90 16.5 (Dead) 4. 61/M(#179918) 700 40/60 8.5 (Dead) 5. 47/M (#171024) 350 60/70 6 (Dead) * 6.73/M (#185507) 400 40/80 9 (Dead) 7. 68/M (#185938) 600 60/90 6.5 (Dead)8. 69/M (#189953) 600 50/60 8 (Dead) 9. 56/M/NSCC 600 40/70 7.5 (Dead)10. 65/M/ 380 50/60 5.5 (Dead) Median: 68.0 yrs 575 45/70 8.25 *Karnofsky Performance Status ** Survival measured from date of firsttreatment

TABLE 7 Complications of SEC Treatment Complication (no. of patients)Fever >38° C. 6 Chills 2 Pain 3 Dyspnea 0 Leukopenia 0 No toxicity 6

Toxicity

Adverse effects associated with SSEC treatment are shown in Table 7. Ingeneral the SEC was well tolerated. The most common adverse effect wasfever ranging from 37.4°-39.8° C. that was unrelated to dosage. Feverreached 38° C. in 5 patients and 39.8° C. in 6 and lasted for 24-36hours. However in 2 patients, it lasted more than 36 hours and wasrelieved by indomethacin suppository. Minimal pleuritic chest painipsilateral to the effusion occurred in 3 patients and abatedspontaneously. There was no evidence of respiratory distress, congestiveheart failure and no significant changes in hepatic or renal functionduring or after treatment. No stage 3 or 4 ltoxicity was observed in anycase.

Hematologic Changes in the Blood and Pleural Fluid

Peripheral blood white blood cell, neutrophil and lymphocyte countsincreased slightly but consistently six hours after treatment in allpatients studied (see Table 8. Likewise, total leukocyte, neutrophil andlymphocyte counts in pleural effusions increased significantly 6 hoursafter treatment.

TABLE 8 Hematologic Changes in Blood and Effusions of Patients Treatedwith Intrapleural SEC WBC Neutrophil Lymphocytes (10⁹/L) (10⁹/L) (10⁹/L)Peripheral Blood Pre-treatment 5.200 ± 0.398 3.285 ± 2.50 1.850 ± 0.144Post-treatment 8.533 ± 1.534* 6.455 ± 1.535* 1.916 ± 0.587 PleuralEffusion Pre-treatment 0.761 ± 0.150 0.553 ± 0.150 0.201 ± 0.134Post-treatment 1.178 ± 0.381* 0.661 ± 0.185* 0.541 ± 0.167* *significantat p < 0.05

Peripheral blood and pleural effusion leukocyte counts before and 6-24hour after the initial SSEC treatment. Significant increases inleukocyte and neutrophil counts were noted in blood and pleural fluid.Lymphocyte counts were significantly elevated post-treatment only in thepleural fluid.

Discussion

In general, these results for survival and responses (control of pleuraleffusion) suggest that SEC was not only palliating the symptoms of MPEbut also exerted a therapeutic effect on the patient's tumor. Thisoccurred in the absence of significant clinical or serologic toxicity.These results show that intrapleural administration of SEC in patientswith MPE can eliminate pleural fluid reaccumulation for more than 90days and, in several cases, for as long as 6, 8, 12 and 15 months. Theresponse rate (100%) for clearance of MPE exceeded that for agents nowin common use for palliation of MPEs, namely talc, bleomycin anddoxycycline.

A substantial number of the patients receiving SEC treatment survivedlonger than would be expected than if the SEC were only palliative. TheMST of 8.25 months in the 10 NSCLC patients included 3 patients whosurvived more than 350 days. At the time these results were lastanalyzed, one patient was still alive 26 months after startingtreatment. In contrast, 21 patients with MPE from NSCLC who were treatedwith talc pleurodesis at the Univ. of California, San Diego (UCSD) from1993-1998 showed a MST of 2.5 months (Burroughs et al., Chest 717:73-78(2000)). Eleven patients from the latter group with comparablepretreatment KPS to the SEC-treated group had a median survival of 2.4months. Both groups had a statistically similar distribution of KPS yetthe SEC-treated group had a median survival on the order of 3 foldlonger than controls and a 1 year survival of 20% compared to 0% forcontrols.

An additional group of 61 patients with MPE from NSCLC treated withcatheter drainage and chemical pleurodesis at M.D. Anderson CancerCenter from 1994-1996 had a median survival of 2.0 months (Putnam etal., Ann. Thorac. Surg. 69: 369-375 (2000). The improved survival in theSEC group could not be attributed to higher pretreatment KPS scoressince the median pre-treatment KPS for the SEC-treated 10 patients was45 compared to >70 in the palliative-treatment control group. Despitethe lower pretreatment KPS, the SEC-treated group had an extension oftheir MST on the order of 4 fold compared to controls. Hence, comparedto current historical controls treated with the best availablepalliative regimens (talc, doxycycline, catheter drainage), theSEC-treated patients of the present study survived significantly longer.

Moreover, as noted by others, initial pleural fluid volume was notpredictive of improved survival. Indeed, the longest survivor had onlythe fifth largest initial pleural effusion.

The observed SEC-induced MST of 8.25 months (a) exceeded the 7.6 monthMST observed in a study of 262 stage 1V lung cancer patients receivingthe best single agent chemotherapy (cisplatin) and (b) was comparable tothe 8.6 month MST in patients receiving the best combinationchemotherapy (cisplatin gemcitabine).

In contrast to talc, bleomycin, doxycycline and catheter drainage, SECtreatment does not require thoracotomy, chest tube insertion orin-hospital tube drainage. The SEC treatment was performed in the officeby thoracentesis followed by instillation of SEC into the pleural space.In general, SEC treatment induced minimal side effects and toxicity. Inparticular, there was no dyspnea, pulmonary edema or acute respiratorydistress syndrome as has been observed rarely following talcinsufflation or instillation. As a safe outpatient procedure, SECtherapy appears to offer considerable cost reduction compared to thepresently available agents which while also providing symptomatic reliefand a significant survival benefit. Table 9 shows the comparative costeffectiveness with palliative treatments.

TABLE 9 AGENT COST OF TOTAL TREATMENT TREATMENT COST COST DRIVERS Talcinsufflation $0.15-0.50 $30,996 OR Facilities, Thoracic Surgeon, (2.5-10g) Respiratory Therapy, Hospitalization, Indwelling Chest Tube,Complications (ARDS) Talc slurry $0.15-0.50 $25,000 Hospital days,Respiratory Therapy, (2.5-10 g) Indwelling Chest Tube, ComplicationsBleomycin $1104 $20,000 High Agent cost, Hospitaiization, IndwellingChest Tube, Toxicity Potential with Chemotherapy Low Response Rate, HighRecurrence Rate SEC  $300 $2000-$10,000 NONE of the following: ORfacility Superantigen Thoracic Surgeon, Hospitalization, RespiratoryTherapy, Indwelling Chest Tube & Drainage,

Patients with MPEs from small cell carcinoma of the lung, uterinesarcoma and melanoma were treated with intrapleural SEC and showedresolution of their MPEs for 1.5-4 months. Thus, it would be expectedthat SEC therapy is applicable to a substantial number of malignantpleural effusions from tumors other than NSCLC.

While SEC therapy is effective against symptomatic MPEs as given above,it is also applicable to patients with small asymptomatic malignantpleural effusions irrespective of origin or initial pleural fluidvolume. In lung cancer in particular, the presence of a malignantpleural effusion portends a prognosis of two months survival(irrespective of initial effusion volume). Thus, small symptomatic orasymptomatic MPEs originating from lung, breast, stomach, esophagus,colon, kidneys, ovary, uterus (or any other origin) as well as melanoma,lymphomas and mesotheliomas would be expected to benefit from thistreatment which will prolong survival in these groups.

Example 2 Treatment of Lung Adenocarcinoma by Intratumoral Injection ofSEA Followed by Intratumoral Chemotherapy Patient and Treatment Plan

The patient is a 75 year old man with a large adenocarcinoma in the leftmidlung field. He received intratumoral administration of SEC (5 ng)once weekly for 7 weeks. During weeks 8-11, the patient received weeklyintratumoral injections of SEA (5 ng) together with cisplatin (10 mg).Chest x-rays were done before treatment and 1 week after the conclusionof the last dose of intratumoral SEC/cisplatin.

Criteria for response are as set forth by the International UnionAgainst Cancer and are given in more detail below. Briefly, a completeresponse is defined as no measurable disease. A partial response isdefined as a 50% reduction of the bidirectional diameter of measurabletumor.

Results: One week after concluding the course of intratumoral SEACfollowed by intratumoral SEA+cisplatin, the patient's chest x-ray and CTscan showed complete disappearance of the pulmonary nodule whichmeasured 20 cm³ before commencing treatment. The lesion showedprogressive reduction in size on ultrasound during the SEA treatmentphase. Morbidity consisted of a low grade temperature for 3-4 weeksafter commencing SEC therapy, fatigue and anorexia not requiringtreatment. These symptoms abated with continued treatment. CBC, renaland liver functions tests did not change significantly after treatments.Discussion: A SAg administered alone intratumorally for 7 weeks followedby a 3 week course of a combination of the SAg and low dose cisplatin,given intratumorally, induced complete remission. The dose of cisplatinused is more than 10-fold lower than the mean recommended doseadministered systemically per cycle. Side effects of the SEA treatmentwere minimal, and cisplatin caused no toxicity. This patientsubsequently received two cycles of systemic cisplatin and mitomycin Cand remains in complete remission 7 months later.

Example 3 Clinical Trial of Intratumoral SAg and Low Dose Chemotherapyin Humans Patients

All patients treated have histologically confirmed malignant massesconfirmed by biopsy or cytology are entered. Malignant diseasesincluding carcinomas, sarcomas, melanomas, gliomas neuroblastomas,lymphomas and leukemia. The malignant disease has failed to respond oris advancing despite conventional therapy. Patients in all stages ofmalignant disease involving any organ system are included. Stagingdescribes both tumor and host, including organ of origin of the tumor,histologic type, histologic grade, extent of tumor size, site ofmetastases and functional status of the patient. For a generalclassification includes the known ranges of Stage 1 (localized disease)to Stage 4 (widespread metastases), see Abraham J et al., BethesdaHandbook of Clinical Oncology, Lippincott, Williams & Wilkins,Philadelphia, Pa., 2001. Patient history is obtained and physicalexamination performed along with conventional tests of cardiovascularand pulmonary function and appropriate radiologic procedures. Themalignant masses are visible on x-ray or CT scan and are measurable withcalipers. They have not been undergoing any other anticancer treatmentfor at least one month and have a clinical KPS of at least 50.

SEA is used as the prototypical SAg (but other SAgs and homologues asdescribed herein are used in other patients in comparable doses,yielding similar results). SEA is administered intratumorally in dosesof 0.05-1 ng/kg intratumorally once every 2-7 days. The tumors areinjected under direct vision at surgery, bronchoscopy, endoscopy,peritoneoscopy, culdocopy. They are accessible to percutaneous injectionwith CT, ultrasound or stereotaxis used to localize and guide theinjected composition into the tumor.

Intratumoral chemotherapy preferably comprises the use of a selectedsingle agent which is known in the art to be effective against aparticular tumor. Intratumoral combination chemotherapy wherein eachagent is given in a reduced dose can also be used. Total intratumoraldose of a chemotherapeutic agent per cycle is 3-7 fold below that of themean recommended close of a systemic chemotherapeutic agent per cycle.Recommended mean dosages for single and individual chemotherapeuticagents for human tumors are well known in the art and given in Abrahamet al., supra. The intratumoral dose of a cytotoxic drug administered tothe tumor site generally ranges from about 0.1 to 500, more usuallyabout 0.5 to 300 mg/kg body weight, depending upon the nature of thedrug, size of tumor, and other considerations. The intratumoralchemotherapy is given after at least 2-7 weekly of intratumoral SAginjections and within 36 hours after the previous SAg treatment. The SAgand chemotherapy are given at the same time and continued every 7 daysfor at least 3 treatments and up to 6 weekly treatments if the tumor isshrinking and the there is no dose limiting toxicity.

Systemic chemotherapy is also used in doses 10-95% below the meanrecommended therapeutic dose for a single agent alone or in combinationwith other chemotherapeutic agents. While a range of 10-95% reduction isuseful, chemotherapeutic closes 50% below the recommended mean dose percycle are used most often. Systemic or intratumoral chemotherapy is alsostarted after the first SAg treatment but is usually given within 36hours after 2-7 intratumoral SEA treatments. The SEA and chemotherapy isalso given at the same time beginning with the first treatment butgenerally after 2-7 SEA treatments. The intratumoral chemotherapy iscontinued alone or together with intratumoral SEA for at least 3 weeklyinjections after at least 2-7 intratumoral treatments with SEA alone Itis continued for an additional 3-6 weeks if the tumor is shrinking andthere is no dose limiting toxicity.

In the case of a lung tumor, a typical treatment consists ofpercutaneous or transbronchial injection of a lung tumor noduleintratumorally with SEA 5 ng every 7 days for 7 weeks followed by SEA 5ng with cisplatin 10 mg intrattunorally every 7 days for three weeks.The chemotherapy is used alone (i.e. without the SEA) or together withSEA for the last three treatments. For large tumors exceeding 40 cm²(two dimensions), injections are given at more than one site in thetumor mass using doses that cumulatively do not exceed that of a singledose per cycle. Likewise, additional malignant nodules or masses aretreated in the same fashion as large single nodules. Alternatively,additional nodules are treated sequentially following the completion ofone cycle in a single mass.

Representative doses of single agent chemotherapeutic agents used in anaverage sized adult for intratumoral injection against the more commontumors are, (1) Breast carcinomas: Doxorubicin (14-30 mg/treatment ×3),Taxol (30 mg/treatment ×3); (2) Colo-rectal cancer: 5-Fluorouricil (180200 mg/treatment ×3); Lung cancer: cisplatin (4-10 mg/treatment ×3). Thedrugs are administered intratumorally in 1 ml normal saline over a 1minute period.

Patient Evaluation: Assessment of response of the tumor to the therapyis made once per week during therapy and 30 days thereafter using CT orx-ray visualization. Depending on the response to treatment, sideeffects, and the health status of the patient, treatment is terminatedor prolonged from the standard protocol given above. Tumor responsecriteria are those established by the WHO and RECIST (ResponseEvaluation Criteria in Solid Tumors) summarized below (also Abraham etat., supra)

RESPONSE DEFINITION Complete remission (CR) Disappearance of allevidence of disease Partial remission (PR) □50% decrease in the productof the two greatest perpendicular tumor diameters; no new lesions Lessthan partial 25%-50% decrease in tumor size, stable remission (<PR) forat least 1 month Stable disease <25% reduction in tumor size; noprogression or new lesions Progression ≧25% increase in size of any onemeasured lesion or appearance of new lesions despite stabilization orremission of disease in other measured sites

The efficacy of the therapy in a patient population is evaluated usingconventional statistical methods, including, for example, the Chi Squaretest or Fisher's exact test. Long-term changes in and short term changesin measurements are evaluated separately.

Results

A total of 810 patients are patients treated. The number of patients foreach tumor type and the results of treatment are summarized in Table 10.Positive tumor responses are observed in as high as 80-90%% of thepatients with breast, gastrointestinal, lung, prostate, renal andbladder tumors as well as melanoma and neuroblastoma as follows:

Six hundred and sixty five patients with all tumors exhibit objectiveclinical responses for an overall response rate of 82%. Tumors generallystart to diminish and objective remissions are evident after four weeksof combined SEA-chemotherapy. Responses endure for an average of 24months.

Toxicity consists of mild short-lived fever, fatigue and anorexia notrequiring treatment. The incidence of side effects (as % of totaltreatments) are as follows: chills—10; fever—10; pain—5; nausea—5;respiratory—3; headache—3; tachycardia—2; vomiting—2; hypertension—2;hypotension—2; joint pain—2; rash—2; flushing—1; diarrhea—1;itching/hives—1; bloody nose—1; dizziness—<1; cramps—<1; fatigue—<1;feeling faint—<1; twitching—<1; blurred vision—<1; gastritis<1; rednesson hand—<1. Fever and chills are the most common side effects observed.Side effects are somewhat less frequent inpatients treated withintratumoral SAg plus low dose single agent chemotherapy compared withSAg and full dose systemic chemotherapy. Side effects are less prevalentwith the intratumoral SAg-chemotherapy regimen compared with SAg andfull dose systemic chemotherapy regimen but this is not statisticallydifferent. CBC, renal and liver functions tests do not changesignificantly after treatments.

TABLE 10 All Patients % of Patients No. Response Responding 567 CR 70 70PR 8.6 28 <PR   3.4 By Tumor Type: Breast adenocarcinoma 100 CR + PR +<PR 80% Gastrointestinal carcinoma 100 CR + PR + <PR 85% Lung Carcinoma150 CR + PR + <PR 90% Brain glioma/astrocytoma 50 CR + PR + <PR 80%Prostate Carcinoma 100 CR + PR + <PR 80% Lymphoma/Leukemia 80 CR + PR +<PR 75% Head and Neck Cancer 80 CR + PR + <PR 75% Renal and BladderCancer 50 CR + PR + <PR 90% Melanoma 50 CR + PR + <PR 80% Neuroblastoma50 CR + PR + <PR 80%

Example 4 Treatment Plan and Outcome Prediction using IntrapleuralSuperantigens

Patients have with malignant pleural effusions confirmed by biopsy orpleural fluid cytology and have not been undergoing any other anticancertreatment for at least one month and have a clinical Karnofsky status ofat least 60-70%. SEA in doses of 10-30 nanograms is administeredintrapleurally once or twice weekly immediately after drainage of theeffusion via conventional thoracentesis. This procedure is performedonce or twice weekly in an outpatient or office setting. Treatment iscontinued once weekly until effusion does not recur. An objectiveresponse is recognized as no reaccumulation of pleural fluid 30 daysafter treatment (DeCamp M M et al., Chest 112: 291S-295S (1997); FentonK N et al., Am J. Surg. 170: 69-74 (1995)).

There are 90 evaluable patients with malignant pleural effusions treatedwith intrapleural SEA. All patients have stage IIIb or stage IV lungcancer. There are 50 evaluable patients with malignant ascites. Eightyfive patients with pleural effusions exhibit objective clinicalresponses for a response rate of 94.5%. Effusion reaccumulation (atweekly intervals) progressively diminished after each SEA treatment. Anexample of progressive reduction of effusion reaccumulation after eachtreatment is shown below. Patients required an average of threetreatments before there was no significant reaccumulation. However,several patients required only one treatment to eliminate fluidreaccumulation. Forty five patients with malignant ascites showobjective responses for a response rate of 90%.

Toxicity in both malignant pleural effusion and ascites consists of mildshort-lived fever, fatigue and anorexia not requiring treatment. CBC,renal and liver functions tests did not change significantly aftertreatments.

The SAg has better therapeutic efficacy for malignant pleural effusionsand ascites than existing agents (talc, bleomycin, doxycycline) withoutthe discomfort and complications associated with an indwelling drainingchest tube. In the case of pleural effusion, It is also 90% morecost-effective compared to existing therapy since it is carried out inan outpatient facility and does not involve the major costs associatedwith hospitalization, i.e., chest tube insertion, operating and recoveryroom, indwelling chest tube drainage and respiratory therapy.

Example 5

Anti-Tumor Effects of Intratumoral SA and Chemotherapeutic AgentsAdministered in Viscous Form of Controlled Release Formulation

The SAgs and chemotherapeutic agents are prepared in controlled releaseformulations. The preparation of the preferred biodegradable controlledrelease formulation for intratumoral administration of SAg and cisplatinas a preferred single agent for use in patients with NSCLC is described.Cisplatin is a representative chemotherapeutic agent; otherchemotherapeutic agents preferred for a given type of tumor be preparedan used similarly with slight variations that are within the skill ofthe art.

Cisplatin for Injection, USP (Platinol®, 10 mg vial) manufactured byBristol Laboratories or lyophilized CDDP manufactured by Faulding (DavidBull Laboratories, Australia) is used. Aqueous collagen gel, 6.5% isobtained from Collagen Corporation (Palo Alto, Calif.), 0.3 ml nominalfill in 1 ml plastic syringes. The gel comprises a highly purified,telopeptide-free bovine Type I collagen, 6.5% (w/w); sodium phosphates,0.1 M; sodium chloride, 0.045 M; and has a nominal pH of 7.2. Optionallyepinephrine is used as a solution (1 mg/ml). Polysorbate 80 is obtainedfrom PPG Industries. Carboxymethylcellulose sodium (NaCMC) is obtainedfrom Aqualon. 0.9% Sodium Chloride for injection, USP (10 ml vial)(“saline”) and sterile water for injection (‘WFI’), USP (10 ml vial) maybe obtained from common sources (e.g., Abbott Laboratories).

Diluent contain polysorbate 80, 1.0 mg; EDTA disodium dihydrate, 0.1 mg;USP carboxymethylcellulose 0.5 mg; sodium metabisulfite, 0.2 mg; glacialacetic acid USP, 0.49 mg; sodium acetate, anhydrous, 0.15 mg; WFI up to1 ml. HCl and/or NaOH may be added if necessary, to adjust pH to 4.0.USP epinephrine, 0.160 mg is also optional. A second diluent containsall of the above ingredients except for carboxymethylcellulose.

The viscous form of Cisplatin is prepared by diluting 5-10 mg in 1-4 ccof diluent. The resulting solution is very viscous and can serve as acontrolled release formulation upon injection into tumor. This is thepreferred method of administration of this drug. SAg can be present inthe same solution as cisplatin as a viscous mixture. In this way Bothcisplatin and SAg can be injected into the tumor at the same time.

The gel is prepared by combining the various components in a sterileenvironment. Upon admixture of the bovine collagen matrix and otheragents, a uniform dispersion is obtained. For collagen and collagenderivatives, the material is provided as a uniform dispersion ofcollagen fibrils in an aqueous medium, where the collagenous materialranges in concentration from about 5 mg/ml to not more than about 100mg/ml. The drug and or SAg may then be added to the collagenousdispersion using agitation to ensure uniform dispersion of the activeagents. Other materials, as appropriate, may be added concomitantly orsequentially. After ensuring the uniform dispersion of the variouscomponents in the mixture, the mixture is sterilized and sealed inappropriate containers.

Vials containing either 10 mg or 25 mg of lyophilized CDDP arereconstituted by adding either 1.6 ml or 4.0 ml of diluent,respectively, to yield a suspension of CDDP. SEA is similarlyreconstituted in sterile saline and added in desired concentration tothe CDDP solution. Gels containing CDDP/SEA are prepared in finalvolumes of 2.0 ml or 5.0 ml. Final gels contained 4.0 mg/ml CDDP/5 ngSEA and 0.1 mg/ml of epinephrine, in a 2% collagen matrix, ready foruse.

Intratumoral Therapy: Therapy preferably comprises the use of a selectedsingle agent ((chemo- or biotherapeutic) which is known in the art to beeffective against a particular tumor, e.g., cisplatin/carboplatin forNSCLC, doxorubicin/taxotere for breast carcinoma, 5-Fluoruricil forcolorectal carcinoma, etc. Intratamoral combination chemotherapy whereineach agent is given in a reduced dose are also used. The intratumoralinjection of SEA, 0.05-0.5 ng/kg, is given once weekly for 2-7 weeks.The chemotherapy is started within 36 hours of the last dose of SEA andthen every 7 days for 3 treatments. SEA can also be given together withthe chemotherapy or beginning with the first injection or secondinjection of chemotherapy.

The dose of a chemotherapeutic drug or biologic agent used forintratumoral administration, is reduced 10- to 50-fold below the meanFDA-recommended dose for parenteral administration in a single cycle.Chemotherapeutic concentrations in the sustained release preparationrange from 0.01 to 50 mg/ml. Chemotherapy is given within 36 hours afterthe 7^(th) intratumoral SEA injection and continued once weekly for atleast three weeks. It is extended to six or more weeks if the tumor isdiminishing in size and there is no dose limiting toxicity. Injection ofthe dose is given at more than one site in tumors exceeding 40 cm². Inthis case the dose is divided into two or more portions with thecumulative dose per treatment not to exceed that for a single site fulldose.

Illustrative of the manner of sustained administration is intratumoraladministration of cis-diaminodichloroplatinum (CDDP) in controlledrelease formulation for which the recommended intratumoral dose perweekly injection is 0.05-0.1 mg/kg with a total dose range dose of 12-30mg per cycle. SEA is administered intratumorally in doses of 0.05-1ng/kg intratumorally once weekly for 2-7 weeks followed by CDDP (4-10mg) weekly for 3 weeks. The tumors are accessed via percutaneousinjection using CT, ultrasound or stereotaxis to localize and guide theinjected composition into the tumor. In certain instances, the tumorsare injected under direct vision at surgery, or via bronchoscopy,thoracoscopy, endoscopy, peritoneoscopy, or culdocopy.

TABLE 11 All Patients % of Patients No. Response Responding 657 CR 80 23PR 3 8 <PR   1 By Tumor Type: Breast adenocarcinoma 150 CR + PR + <PR90% Gastrointestinal carcinoma 150 CR + PR + <PR 90% Lung Carcinoma 150CR + PR + <PR 95% Brain glioma/astrocytoma 50 CR + PR + <PR 85% ProstateCarcinoma 100 CR + PR + <PR 85% Lymphoma/Leukemia 80 CR + PR + <PR 80%Head and Neck Cancer 80 CR + PR + <PR 80% Renal and Bladder Cancer 50CR + PR + <PR 95% Melanoma 50 CR + PR + <PR 85% Neurobiastoma 50 CR +PR + <PR 85%Results: A total of 910 patients are patients treated. The number ofpatients for each tumor type and the results of treatment are summarizedin Table 11. Positive tumor responses are observed in as high as 85-95%of the patients with breast, gastrointestinal, lung, prostate, renal andbladder tumors as well as melanoma and neuroblastoma as follows.

Seven hundred and seventy three patients of 910 entered with all tumorsexhibit objective clinical responses for an overall response rate of84%. Tumors generally start to diminish and objective remissions areevident after four weeks of combined SEA-chemotherapy. Responses endurefor an mean of 36 months.

Toxicity consists of mild short-lived fever, fatigue and anorexia notrequiring treatment. The incidence of side effects (as % of totaltreatments) are as follows: chills—12; fever—15; pain—6; nausea—3;respiratory—2; headache—2; tachycardia—4; vomiting—4; hypertension—1;hypotension—2; joint pain—3; rash—1; flushing—4; diarrhea—2;itching/hives—1; bloody nose—1; dizziness—<1; cramps—<1; fatigue—<1;feeling faint—<1; twitching—<1; blurred vision—<1; gastritis<1; rednesson hand—<1. Fever and chills are the most common side effects observed.Toxic effects usually associated with systemically administeredchemotherapeutic agents were not observed. For example, neurotoxicity,hematologic toxicity, and ototoxicity associated with systemicallyadministered cisplatin were not observed. The bone marrow depressioncommonly observed with parenterally administered chemotherapy such asantimetabolites, e.g., 5-Fluorouricil, Methotrexate; alkylating agents,e.g., cyclophosphamide, Ifosamide; tumor antimicrobials, e.g.,doxorubicin, mitomycin C; plant alkaloids, e.g., Taxol, Taxotere; otheragents, e.g., Cisplatin, Carboplatin, Irinotecan 5-Fluorouricil, Taxol,Taxotere is not seen with intratumoral administration of the theseagents in controlled release formulation. Unique toxicities of singleagents such as cardiomyopathy with the anthracycline antibioticsDoxorubicin, Daunorubicin, hemorrhagic cystitis with Cyclophosphamideand Ifosamide, neurotoxicity with 5-Fluorouricil, neuropathy andarrythmias with Taxol, severe diarrhea with Irinotecan, interstitialpneumonia and hemolytic-uremic syndrome with Mitomycin C are notobserved when administered intratumorally as controlled releaseformulations. Of note, with all intratumoral chemotherapy in this form,there is minimal hematologic toxicity of the intratumorally administeredchemotherapeutic agents and no significant renal and liver toxicity.

All the references cited above are all incorporated by reference herein,whether specifically incorporated or not.

In addition, the following co-pending patent applications areincorporated by reference in their entirety:

Inventor Serial No. Filing Date Title Terman, D. S. 60/438,686 Jan. 9,2003 Intrathecal and Intrapleural Superantigens to Treat MalignantDisease Terman, D. S. 60/415,310 Oct. 1, 2002 Intrathecal andIntratumoral Superantigens to Treat Malignant Disease. Terman, D. S.60/406,750 Aug. 29, 2002 Intrathecal Superantigens to Treat MalignantFluid Accumulation Terman, D. S. 60/415,400 Oct. 2, 2002 Composition andMethods for Treatment of Neoplastic Diseases Terman, D. S. 60/406,697Aug. 28, 2002 Compositions and Methods for Treatment of NeoplasticDiseases Terman, D. S. 60/389,366 Jun. 15, 2002 Compositions and Methodsfor Treatment of Neoplastic Diseases Terman, D. S. 60/378,988 May 8,2002 Compositions and Methods for Treatment of Neoplastic DiseasesTerman, D. S. 09/870,759 May 30, 2001 Compositions and Methods forTreatment of Neoplastic Diseases Terman, D. S. 09/751,708 Dec. 28, 2000Compositions and Methods for Treatment of Neoplastic Diseases

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

1. A method of treating a subject with a primary or metastatic carcinomaof the lung or pleura with or without pleural effusion comprisingadministering to said subject parenterally by infusion or injection atumoricidally effective amount of a composition consisting of: (i) anative staphylococcal enterotoxin or streptococcal pyrogenic exotoxinprotein which native protein: (a) has the biological activity ofstimulating T cell mitogenesis via a T cell receptor νβ region or (ii) abiologically active homologue or fragment of a native staphylococcalenterotoxin or streptococcal pyrogenic exotoxin, which homologue orfragment: (a) has the biological activity of stimulating T cellmitogenesis via a T cell receptor νβ region and (b) has sequencehomology characterized as a z value exceeding 13 when the sequence ofthe homologue or said fragment is compared to the sequence of a nativestaphylococcal enterotoxin or a native streptococcal pyrogenic exotoxin,determined by FASTA analysis using gap penalties of −12 and −2, Blosum50 matrix and Swiss-PROT or PIR database; or (iii) a biologically activefusion protein having said biological activity and said sequencehomology, comprising (A) said homologue, (B) a native staphylococcalenterotoxin, (C) a native streptococcal pyrogenic exotoxin, or (D) abiologically active fragment of said homologue, said native enterotoxinor said native exotoxin, fused to a peptide or polypeptide fusionpartner.
 2. A method of treating a subject with a primary or metastaticcarcinoma of the lung or pleura with or without pleural effusioncomprising administering to said subject parenterally by infusion orinjection a tumoricidally effective amount of a composition consistingof (i) a native staphylococcal enterotoxin or streptococcal pyrogenicexotoxin protein which native protein: (a) has the biological activityof stimulating T cell mitogenesis via a T cell receptor νβ region or(ii) a biologically active homologue or fragment of a nativestaphylococcal enterotoxin or streptococcal pyrogenic exotoxin, whichhomologue or fragment: (a) has the biological activity of stimulating Tcell mitogenesis via a T cell receptor νβ region and (b) has sequencehomology characterized as a z value exceeding 13 when the sequence ofthe homologue or said fragment is compared to the sequence of a nativestaphylococcal enterotoxin or a native streptococcal pyrogenic exotoxin,determined by FASTA analysis using gap penalties of −12 and −2, Blosum50 matrix and Swiss-PROT or PIR database; or (iii) a biologically activefusion protein having said biological activity and said sequencehomology, comprising (A) said homologue, (B) a native staphylococcalenterotoxin, (C) a native streptococcal pyrogenic exotoxin, or (D) abiologically active fragment of said homologue, said native enterotoxinor said native exotoxin, fused to a peptide or polypeptide fusionpartner, wherein a chemotherapeutic drug or drugs is/are adminsteredparenterally by infusion or injection before, together with or afteradministration of said enterotoxin or exotoxin composition.
 3. A methodof treating a subject with a primary or metastatic carcinoma of the lungor pleura with or without pleural effusion comprising administering tosaid subject parenterally by infusion or injection a tumoricidallyeffective amount of a composition consisting of: (i) a nativestaphylococcal enterotoxin or streptococcal pyrogenic exotoxin proteinwhich native protein: (a) has the biological activity of stimulating Tcell mitogenesis via a T cell receptor νβ region or (ii) a biologicallyactive homologue or fragment of a native staphylococcal enterotoxin orstreptococcal pyrogenic exotoxin, which homologue or fragment: (a) hasthe biological activity of stimulating T cell mitogenesis via a T cellreceptor νβ region and (b) has sequence homology characterized as a zvalue exceeding 13 when the sequence of the homologue or said fragmentis compared to the sequence of a native staphylococcal enterotoxin or anative streptococcal pyrogenic exotoxin, determined by PASTA analysisusing gap penalties of −12 and −2, Blosum 50 matrix and Swiss-PROT orPIR database; or (iii) a biologically active fusion protein having saidbiological activity and said sequence homology, comprising (A) saidhomologue, (B) a native staphylococcal enterotoxin, (C) a nativestreptococcal pyrogenic exotoxin, or (D) a biologically active fragmentof said homologue, said native enterotoxin or said native exotoxin,fused to a peptide or polypeptide fusion partner, wherein a therapeuticclose of x-irradiation is administered before, together with or afteradministration of said enterotoxin or exotoxin composition.
 4. A methodof treating a subject with a primary or metastatic carcinoma comprisingadministering intratumorally or intrathecally by infusion or injectionto said subject a tumoricidally effective amount of a compositionconsisting of (i) a native staphylococcal enterotoxin or streptococcalpyrogenic exotoxin protein which native protein: (a) has the biologicalactivity of stimulating T cell mitogenesis via a T cell receptor νβregion or (ii) a biologically active homologue or fragment of a nativestaphylococcal enterotoxin or streptococcal pyrogenic exotoxin, whichhomologue or fragment: (a) has the biological activity of stimulating Tcell mitogenesis via a T cell receptor νβ region and (b) has sequencehomology characterized as a z value exceeding 13 when the sequence ofthe homologue or said fragment is compared to the sequence of a nativestaphylococcal enterotoxin or a native streptococcal pyrogenic exotoxin,determined by FASTA analysis using gap penalties of −12 and −2, Blosum50 matrix and Swiss-PROT or PIR database; or (iii) a biologically activefusion protein having said biological activity and said sequencehomology, comprising (A) said homologue, (B) a native staphylococcalenterotoxin, (C) a native streptococcal pyrogenic exotoxin, or (D) abiologically active fragment of said homologue, said native enterotoxinor said native exotoxin, fused to a peptide or polypeptide fusionpartner.
 5. The method of claims 1, 2, 3 and 4 wherein the fusionpartner is an antibody or antibody fragment specific for tumor cells ortumor vasculature expressing erb/neu, MUC1, 5T4, endoglin, TGFβ.receptor, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a VEGF/VPFreceptor, a FGF receptor, a TIE, an α_(ν)β₃ integrin, a pleiotropin, anendosialin, cytokine-inducible or coagulant-inducible products ofintratumoral blood vessels, aminophospholipids, phosphatidylserine orphosphatidylethanolamine or a polypeptide capable of directly orindirectly stimulating coagulation is a truncated tissue factor thatactivates Factor X in the presence of Factor VIIa but fails to bind tophospholipid membranes and convert Factor VII to Factor VIIa.
 6. Themethod of claims 1, 2, 3 and 4 wherein the native staphylococcalenterotoxin and streptococcal pyrogenic exotoxin is selected from agroup comprising SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, TSST-1, SEG, SEH,SEI, SEJ, SEK, SEL, SEM, SPEA, SPEB, SPEC, SSA, SPEG, SPEH, SMEZ
 7. Themethod of claims 1, 2, 3 wherein the tumoricidally effective amount ofthe compositions are administered to said subject (i) intravenously,(ii) intramuscularly, (iii) subcutaneously, (iv) intradermally (or (v)by any two or more of routes (i)-(iv).
 8. The method of claim 2 whereinthe chemotherapeutic agent is docetaxel, paclitaxel, taxol, taxotere,cisplatin, doxorubicin, vinorelbine, gemcitabine, camptothecindactinomycin, mitomycin, caminomycin, daunomycin, tamoxifen,vincristine, vinblastine, etoposide, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, actinomycin-D, mitomycinC, aminopterin, combretastatin(s) and derivatives and prodrugs thereof.9. The method of claim 3 wherein x-irradiation to the carcinoma in dosesof 60-65Gy beginning up to 30 days before, at the same time or 30 daysafter the start of said administration of said staphylococcalenterotoxin or streptococcal pyrogenic exotoxin composition.